acid properties  - Crack Key For U

Did you know that the egg yolk contains almost as much protein as the egg white? protein source, because they contain all 9 essential amino acids. A Compilation of Abstracts and Key Word and Author Indexes United States. Activation analysis ; crystal structure ; diffraction ; isotopes ; molecular. Table 1: Characteristics lead acid battery electrolyte (35% H2SO4 / 65% water). Health Risks In case of a small electrolyte spilled, you should.

: Acid properties - Crack Key For U

360 TOTAL SECURITY CRACK ZIP - FREE ACTIVATORS
SCREENHUNTER 6.0 FREE PORTABLE
SUBLIME TEXT 4107 CRACK PRODUCT KEY FULL FREE DOWNLOAD 2021
Acid properties - Crack Key For U

watch the video

ACID Properties in DBMS With Examples - In-depth Explanation

Acid properties - Crack Key For U -

Six-mark questions

Six-mark questions are extended open response questions. These require longer answers than the structured questions. It is wise to plan your answer rather than rushing straight into it, otherwise you may stray away from the key points.

To gain full marks you need to:

  • support explanations using scientific knowledge and understanding
  • use appropriate scientific words and terms
  • write clearly and link ideas in a logical way
  • maintain a sustained line of reasoning, rather than getting lost or bogged down

Six-mark questions often use these command words:

  • describe - you need to give an account but no reason
  • explain - you must give reasons or explanations
  • devise - you must plan or invent a procedure using your scientific knowledge and understanding
  • evaluate - you must review information, including identifying strengths and weaknesses, and make a supported conclusion

Six-mark questions may be synoptic questions. These questions bring together ideas from two or more topics. For example, a question about fertilisers could include ideas about covalent substances, acids and alkalis, chemical calculations and effects on the environment.

The answers shown here give marking points as bullet points. You do not usually need to include all of them to gain six marks but you do need to write in full sentences, linking them logically and clearly.

Edexcel questions courtesy of Pearson Education Ltd.

Sample question 1 - Foundation

Question

Marble chips react with dilute hydrochloric acid to produce carbon dioxide gas.

The rate of this reaction can be changed by changing the size of the marble chips.

Describe how you could investigate what effect using smaller marble chips has on the rate of this reaction. Predict and explain the effect of using smaller marble chips on the reaction rate. [6 marks]

Your answer should include the following:

  • measure volume of acid/stated volume
  • measure mass of marble chips/stated mass
  • add acid to marble or marble to acid in a suitable container, eg flask, beaker, boiling tube, test tube
  • collect the gas in a gas syringe/measuring cylinder over water or bubble gas through limewater/bubble gas through water
  • measure the amount/volume of carbon dioxide or count the bubbles/fixed volume of carbon dioxide
  • measure mass/mass loss (on a balance)
  • time/measure how long the reaction takes
  • repeat experiment with different size marble chips
  • use the same mass of marble chips
  • use the same volume/concentration/mass of acid/same acid
  • crush the marble/use powdered marble

Results:

  • smaller chips (of marble) have a more vigorous
  • reaction/produce more fizzing/bubbles
  • smaller chips take less time to react/produce a certain volume of gas/have a certain mass loss
  • smaller chips have a larger surface area
  • smaller chips react faster
  • larger surface gives a faster reaction

[6]

Sample question 2 - Foundation

Question

Hydrochloric acid reacts with magnesium metal to produce hydrogen gas:

Magnesium + hydrochloric acid → magnesium chloride + hydrogen

Describe how you could use magnesium ribbon, and a solution of hydrochloric acid, to show that decreasing the concentration of the hydrochloric acid changes the rate of this reaction. [6 marks]

Your answer should include the following:

Method:

  • dilute the acid...
  • to make different concentrations
  • add magnesium to acid...
  • in suitable container
  • equal volumes of the acids
  • equal lengths of magnesium

Observations:

  • observe/count bubbles
  • highest concentration magnesium reacts, lowest concentration magnesium does not react
  • time how long it takes for the magnesium to disappear
  • measure volume gas produced
  • measure decrease in mass

Analysis:

  • formed bubbles faster
  • magnesium disappears faster
  • gas produced faster
  • mass lost faster

[6]

Sample question 3 - Higher

Question

A student investigated the rate of reaction between zinc and dilute sulfuric acid:

Zn(s) + H2SO4(aq) → ZnSO4(aq) + H2(g)

The student carried out two experiments, using the same mass of zinc and the same sized pieces of zinc. The results are shown in the table.

Experiment 1Experiment 2
Concentration of sulfuric acid0.5 mol dm-31.5 mol dm-3
Temperature20°C40°C
Rate of reactionSlowFast

Evaluate these results, explaining the reasons why the rate of reaction in Experiment 2 is faster than the rate of reaction in Experiment 1.

In your answer you should refer to the frequency and energy of collisions between particles. [6 marks]

General points:

  • reactions occur when particles collide
  • more frequent collisions cause higher rate of reaction
  • mass and size of zinc pieces stay the same so no effect on rate of reaction
  • because surface area stays the same
  • two factors have been altered in the same experiment
  • cannot be certain of effect of each

Concentration:

  • experiment 2 higher/triple concentration of acid
  • so more particles (in same volume)
  • so more frequent collisions between particles
  • more successful collisions

Temperature:

  • experiment 2 higher temperature
  • particles move faster
  • particles have more energy
  • so more frequent collisions between particles (so increased rate)
  • more successful collisions
  • so more energetic collisions between particles
  • more particles have enough energy to react (activation energy) when they collide

[6]

Sample question 4 - Higher

Question

Reactions can occur when particles collide.

Rates of reactions can be altered by changing conditions.

Explain how the rate of reaction between a solid and a liquid is altered by reducing the size of the pieces of solid and by increasing the temperature of the liquid. [6 marks]

Your answer should include the following:

Smaller pieces of solid:

  • of same mass
  • have larger surface area to volume ratios
  • so more frequent collisions
  • so higher rate of reaction

Higher temperature:

  • particles move faster
  • more frequent collisions
  • particles have more energy
  • more collisions have required energy to react/activation energy
  • more collisions successful
  • higher rate of reaction

[6]

Источник: https://www.bbc.co.uk/bitesize/guides/ztngtv4/revision/6

Acid Rain

Inorganic Reactions Experiment

Authors: Rachel Casiday and Regina Frey
Department of Chemistry, Washington University
St. Louis, MO 63130


Natural Acidity of Rainwater

Pure water has a pH of 7.0 (neutral); however, natural, unpolluted rainwater actually has a pH of about 5.6 (acidic).[Recall from Experiment 1 that pH is a measure of the hydrogen ion (H+) concentration.] The acidity of rainwater comes from the natural presence of three substances (CO2, NO, and SO2) found in the troposphere (the lowest layer of the atmosphere). As is seen in Table I, carbon dioxide (CO2) is present in the greatest concentration and therefore contributes the most to the natural acidity of rainwater.

Gas

Natural Sources

Concentration

Carbon dioxide
CO2
Decomposition 355 ppm
Nitric oxide
NO
Electric discharge 0.01 ppm
Sulfur dioxide
SO2
Volcanic gases 0-0.01 ppm

Table 1

Carbon dioxide, produced in the decomposition of organic material, is the primary source of acidity in unpolluted rainwater.

NOTE: Parts per million (ppm) is a common concentration measure used in environmental chemistry. The formula for ppm is given by:

Carbon dioxide reacts with water to form carbonic acid (Equation 1). Carbonic acid then dissociates to give the hydrogen ion (H+) and the hydrogen carbonate ion (HCO3-) (Equation 2). The ability of H2CO3 to deliver H+ is what classifies this molecule as an acid, thus lowering the pH of a solution.


(1)

 


(2)

Nitric oxide (NO), which also contributes to the natural acidity of rainwater, is formed during lightning storms by the reaction of nitrogen and oxygen, two common atmospheric gases (Equation 3). In air, NO is oxidized to nitrogen dioxide (NO2) (Equation 4), which in turn reacts with water to give nitric acid (HNO3) (Equation 5). This acid dissociates in water to yield hydrogen ions and nitrate ions (NO3-) in a reaction analagous to the dissociation of carbonic acid shown in Equation 2, again lowering the pH of the solution.


(3)

 


(4)

 


(5)

Acidity of Polluted Rainwater

Unfortunately, human industrial activity produces additional acid-forming compounds in far greater quantities than the natural sources of acidity described above. In some areas of the United States, the pH of rainwater can be 3.0 or lower, approximately 1000 times more acidic than normal rainwater. In 1982, the pH of a fog on the West Coast of the United States was measured at 1.8! When rainwater is too acidic, it can cause problems ranging from killing freshwater fish and damaging crops, to eroding buildings and monuments.


Questions on Acidity of Rainwater

1. List two or more ways that you could test the acidity of a sample of rainwater.

2. Write a balanced chemical equation for the dissociation of nitric acid in water. (HINT: Draw an analogy with Equation 2.)

3. The gaseous oxides found in the atmosphere, including CO2 and NO are nonmetal oxides. What would happen to the pH of rainwater if the atmosphere contained metal oxides instead? (HINT: Think back to Experiment 1.) Briefly, explain your answer.


Sources of Excess Acidity in Rainwater

What causes such a dramatic increase in the acidity of rain relative to pure water? The answer lies within the concentrations of nitric oxide and sulfur dioxide in polluted air. As shown in Table II and Figure 1, the concentrations of these oxides are much higher than in clean air.

Gas

Non-Natural Sources

Concentration

Nitric oxide
NO
Internal Combustion0.2 ppm
Sulfur dioxide
SO2
Fossil-fuel Combustion 0.1 - 2.0 ppm

Table II

Humans cause many combustion processes that dramatically increase the concentrations of acid-producing oxides in the atmosphere. Although CO2 is present in a much higher concentration than NO and SO2, CO2 does not form acid to the same extent as the other two gases. Thus, a large increase in the concentration of NO and SO2 significantly affects the pH of rainwater, even though both gases are present at much lower concentration than CO2.

Figure 1

Comparison of the concentrations of NO and SO2 in clean and polluted air.

About one-fourth of the acidity of rain is accounted for by nitric acid (HNO3). In addition to the natural processes that form small amounts of nitric acid in rainwater, high-temperature air combustion, such as occurs in car engines and power plants, produces large amounts of NO gas. This gas then forms nitric acid via Equations 4 and 5. Thus, a process that occurs naturally at levels tolerable by the environment can harm the environment when human activity causes the process (e.g., formation of nitric acid) to occur to a much greater extent.

What about the other 75% of the acidity of rain? Most is accounted for by the presence of sulfuric acid (H2SO4) in rainwater. Although sulfuric acid may be produced naturally in small quantities from biological decay and volcanic activity (Figure 1), it is produced almost entirely by human activity, especially the combustion of sulfur-containing fossil fuels in power plants. When these fossil fuels are burned, the sulfur contained in them reacts with oxygen from the air to form sulfur dioxide (SO2). Combustion of fossil fuels accounts for approximately 80% of the total atmospheric SO2 in the United States. The effects of burning fossil fuels can be dramatic: in contrast to the unpolluted atmospheric SO2 concentration of 0 to 0.01 ppm, polluted urban air can contain 0.1 to 2 ppm SO2, or up to 200 times more SO2! Sulfur dioxide, like the oxides of carbon and nitrogen, reacts with water to form sulfuric acid (Equation 6).


(6)

Sulfuric acid is a strong acid, so it readily dissociates in water, to give an H+ ion and an HSO4- ion (Equation 7). The HSO4- ion may further dissociate to give H+ and SO42- (Equation 8). Thus, the presence of H2SO4 causes the concentration of H+ ions to increase dramatically, and so the pH of the rainwater drops to harmful levels.


(7)

 


(8)


Questions on Sources of Acidity in Rainwater

4. At sea level and 25oC, one mole of air fills a volume of 24.5 liters, and the density of air is 1.22x10-6 g/ml. Compute the mole fraction (i.e.,moles of component /total moles) and molarity of SO2 when the atmospheric concentration of SO2 is 2.0 ppm (see note in Table I).

5.One strategy for limiting the amount of acid pollution in the atmosphere is scrubbing. In particular, calcium oxide (CaO) is injected into the combustion chamber of a power plant, where it reacts with the sulfur dioxide produced, to yield solid calcium sulfite.

a. Write a balanced chemical equation for this reaction. (HINT: Consult the table of common ions in the tutorial assignment for Experiment 1 to view the structure and formula for sulfite; also, use your knowledge of the periodic table to deduce the charge of the calcium ion. Using these facts, you can deduce the formula for calcium sulfite.)

b. Approximately one ton, or 9.0x102 kg, of calcium sulfite is generated each year for every person served by a power plant. How much sulfur dioxide (in moles) is prevented from entering the atmosphere when this much calcium sulfite is generated? Show your calculation.

c. The final stage in the scrubbing process is to treat the combustion gases with a slurry of solid CaO in water, in order to trap any remaining SO2 and convert it to calcium sulfite. A slurry is a thick suspension of an insoluble precipitate in water. Using the solubility guidelines provided in the lab manual for this experiment, predict whether this stage of the scrubbing process will produce a slurry (i.e., precipitate) or a solution (i.e., no precipitate) of calcium sulfite .

d. If MgO, rather than CaO, were used for scrubbing, would the product of the final stage be a slurry or a solution of magnesium sulfite? (Assume that a very large quantity of magnesium sulfite, relative to the amount of water, is produced.)


Environmental Effects of Acid Rain

Acid rain triggers a number of inorganic and biochemical reactions with deleterious environmental effects, making this a growing environmental problem worldwide.

  • Many lakes have become so acidic that fish cannot live in them anymore.
  • Degradation of many soil minerals produces metal ions that are then washed away in the runoff, causing several effects:
    • The release of toxic ions, such as Al3+, into the water supply.
    • The loss of important minerals, such as Ca2+, from the soil, killing trees and damaging crops.
  • Atmospheric pollutants are easily moved by wind currents, so acid-rain effects are felt far from where pollutants are generated.

Stone Buildings and Monuments in Acid Rain

Marble and limestone have long been preferred materials for constructing durable buildings and monuments. The Saint Louis Art Museum, the Parthenon in Greece, the Chicago Field Museum, and the United States Capitol building are all made of these materials. Marble and limestone both consist of calcium carbonate (CaCO3), and differ only in their crystalline structure. Limestone consists of smaller crystals and is more porous than marble; it is used more extensively in buildings. Marble, with its larger crystals and smaller pores, can attain a high polish and is thus preferred for monuments and statues. Although these are recognized as highly durable materials, buildings and outdoor monuments made of marble and limestone are now being gradually eroded away by acid rain.

How does this happen? A chemical reaction (Equation 9) between calcium carbonate and sulfuric acid (the primary acid component of acid rain) results in the dissolution of CaCO3 to give aqueous ions, which in turn are washed away in the water flow.


(9)

This process occurs at the surface of the buildings or monuments; thus acid rain can easily destroy the details on relief work (e.g., the faces on a statue), but generally does not affect the structural integrity of the building. The degree of damage is determined not only by the acidity of the rainwater, but also by the amount of water flow that a region of the surface receives. Regions exposed to direct downpour of acid rain are highly susceptible to erosion, but regions that are more sheltered from water flow (such as under eaves and overhangs of limestone buildings) are much better preserved. The marble columns of the emperors Marcus Aurelius and Trajan, in Rome, provide a striking example: large volumes of rainwater flow directly over certain parts of the columns, which have been badly eroded; other parts are protected by wind effects from this flow, and are in extremely good condition even after nearly 2000 years!

Even those parts of marble and limestone structures that are not themselves eroded can be damaged by this process (Equation 9). When the water dries, it leaves behind the ions that were dissolved in it. When a solution containing calcium and sulfate ions dries, the ions crystallize as CaSO4l 2H2O, which is gypsum. Gypsum is soluble in water, so it is washed away from areas that receive a heavy flow of rain. However, gypsum accumulates in the same sheltered areas that are protected from erosion, and attracts dust, carbon particles, dry-ash, and other dark pollutants. This results in blackening of the surfaces where gypsum accumulates.

An even more serious situation arises when water containing calcium and sulfate ions penetrates the stone's pores. When the water dries, the ions form salt crystals within the pore system. These crystals can disrupt the crystalline arrangement of the atoms in the stone, causing the fundamental structure of the stone to be disturbed. If the crystalline structure is disrupted sufficiently, the stone may actually crack. Thus, porosity is an important factor in determining a stone's durability.


Questions on Effects of Acid Rain

6. Based on the information described above about the calcium ion, and the formula of calcium carbonate (CaCO3), deduce the charge of the carbonate ion. Also, in the structure of the carbonate ion, are any of the oxygens bonded to one another, or all the oxygens bonded to the carbon atom? (HINT: Consult the structure of the common ions given in the tutorial for Experiment 1).

7. In water, H2SO4 can dissociate to yield two H+ ions and one SO42- ion. Write the net ionic equation for the reaction of calcium carbonate and sulfuric acid. (See the introduction to Experiment 2 in the lab manual for a discussion of net ionic equations.)

8. Which is a more durable building material, limestone or marble? Briefly, explain your reasoning.

Additional Links:


References:

Brown, Lemay, and Buster. Chemistry: the Central Science, 7th ed. Upper Saddle River, NJ: Prentice Hall, 1997. p. 673-5.

Charola, A. "Acid Rain Effects on Stone Monuments," J. Chem. Ed.64 (1987), p. 436-7.

Petrucci and Harwood. General Chemistry: Principles and Modern Applications, 7th ed. Upper Saddle River, NJ: Prentice Hall, 1997. p. 614-5.

Walk, M. F. and P.J. Godfrey. "Effects of Acid Deposition on Surface Waters," J. New England Water Works Assn. Dec. 1990, p. 248-251.

Zumdahl, S.. Chem. Principles, 3rd ed. Boston: Houghton Mifflin, 1998. p. 174-6.

Stryer, L. Biochemistry, 4th ed., W.H. Freeman and Co., New York, 1995, p. 332-339.


Acknowledgements:

The authors thank Dewey Holten (Washington University) for many helpful suggestions in the writing of this tutorial.

The development of this tutorial was supported by a grant from the Howard Hughes Medical Institute, through the Undergraduate Biological Sciences Education program, Grant HHMI# 71192-502004 to Washington University.

Copyright 1998, Washington University, All Rights Reserved.

Источник: http://www.chemistry.wustl.edu/~edudev/LabTutorials/Water/FreshWater/acidrain.html

How to heal dry, cracked heels, according to dermatologists

A long winter and spring stuck inside in the dry air may have made the skin on your feet, especially your heels, super dry — dry enough to crack like a fault line. And while COVID-related self-isolation may tempt you to keep your feet hidden, cracks in your heels can fracture into deep cuts, or fissures, that can be pretty painful and even get infected. We asked board certified dermatologists Sheel Desai Solomon, MD, founder of Preston Dermatology & Skin Surgery in North Carolina and Samer Jaber, MD, founder of Washington Square Dermatology in New York City, what causes dry, cracked heels and the best treatment for dry heels.

IN THIS ARTICLE What causes dry, cracked heels What if over-the-counter treatments don't work?

Related

What causes dry, cracked heels

“Cracked heels occur when you have a disruption of your skin barrier,” explains Jaber. “It can be from a medical condition, like psoriasis or eczema, or can occur when your skin is very dry.”

Other variables that can dry skin on heels enough to crack are age (your skin gets thinner, less elastic and some common medications can contribute) and, of course, winter can be quite the culprit. “Heels are at their worst in winter,” says Solomon. “Indoors and outdoors there is less humidity in the air, and a lack of humidity causes the skin to become drier. Drier skin means more cracking and peeling.” This kind of cracking can cause wounds that can easily get infected if not treated, and if you happen to have a compromised immune system or diabetes, an infection can become a serious health risk, Solomon says.

Related

How to care for dry, cracked heels

1. Keep your feet clean and moisturized

Solomon says diligence in keeping your feet clean and moisturized will keep you on the good foot. “Wash feet with non-foaming hydrating cleanser (typically in a cream or milk form) to keep foot skin from drying further and moisturize still-damp feet after every bath or shower,” she recommends. “That's when it’s time to apply products with petrolatum, glycerin, shea butter, vitamin E or jojoba. These ingredients are very effective at preventing moisture loss.” She also recommends a kitchen cabinet remedy to try — honey. “Honey is full of antimicrobial and antibacterial properties great for cleansing and healing wounds, particularly Manuka honey,” she says, saying you can create your own honey foot mask by combining it with a drop of almond oil and slathering it on your heels.

Related

2. Slough off dead skin

To prevent cracks, exfoliation is key. Both experts extol the virtues of moisturizers with exfoliants like urea (not urine, but a similar compound that has been shown to help moisture seep into skin), and salicylic acid, to help prevent heel cracks with regular use. Solomon also recommends using a “safe foot file that doesn’t look like a cheese grater” to remove dead skin. “Using a foot file on your feet after a shower or bath can be a great way to avoid thick calluses or cracks,” she says. “However, if the file has sharp teeth, it is putting you at risk for cuts or scrapes. The goal is to remove the old, dead skin but leave the healthy layer intact to protect [against] infection.”

3. Seal up deep cracks

Cracked heels that have reached the point where they’ve started to bleed can be extremely painful, warns Solomon. Both experts say liquid bandages are an extremely effective way of sealing up cracks to ease the pain of walking on torn skin while keeping the wounds clean.

Related

4. Wear socks made of natural materials

The damp, dark environment of wet socks in shoes and boots can bring on a fungal foot infection that resembles dry skin, warns Solomon. To prevent this from happening, she advises choosing winter socks made from natural materials, such as cotton or wool rather than synthetic blends, to keep your feet sweat and bacteria-free. “Materials like cotton and wool are naturally more absorbent and these moisture-wicking qualities are extremely important during the winter months. An added bonus is that your feet are less likely to smell, too,” she says. Jaber says his trick to beat cracks in severe dry and cold weather is to advise his patients to apply Vaseline onto the heels of their feet before bed and immediately put on white cotton socks to lock in the moisturizer while they sleep.

Related

Treating dry or cracked heels

1. Eucerin Roughness Relief Spot Treatment

Jaber recommends applying this spot treatment to your heels because its formula contains a high percentage of urea and exfoliating alpha hydroxy acid, which are great at removing dead skin cells so the moisturizing components of the formula, like sunflower oil rich in vitamins A and E, can seep in to heal skin.

2. CeraVe Healing Ointment

Dermatologists love this non-comedogenic, lanolin-free formula, because it doesn’t irritate the skin. As it’s almost half petrolatum (qualifying it as an ointment), is enriched with ceramides (to restore your skin’s barrier) and it also contains hyaluronic acid, an ingredient that helps skin retain moisture.

3. CeraVe SA Cream for Rough & Bumpy Skin

Jabar favors this multitasking moisturizer for its bounty of skin-loving ingredients. Lactic, salicylic and hyaluronic acids exfoliate and moisturize; niacinamide, a B vitamin, helps prevent moisture loss; and ceramides 1, 3, and 6-II help restore your skin’s protective barrier. The formula is free of fragrances and dyes and it releases over time to keep your skin super soft.

4. NatraCure 5-Toe Gel Moisturizing Socks

Solomon recommends these socks because they have a liner that uses aloe vera, vitamin E, and shea butter to intensely hydrate your skin, she says. Plus, they are infused with medical-grade mineral oil for an added boost of moisture.

What if over-the-counter treatments don't work?

Both dermatologists say that if the cracks keep up despite proper, frequent application of high-quality over-the-counter products like these, it might be time to have your dermatologist take a peek. “Lack of vitamins, minerals and zinc in your diet can adversely affect your heel health,” Solomon says. “Sometimes there can be a fungus. If it’s just dry skin, dermatologists can prescribe emollients, such as ammonium lactate or urea cream, to get your skin healthy, then switch to a lotion for maintaining that health.”

But the most important thing is to try and determine the cause of the cracking, says Jaber. “If it’s more related to dryness, try and avoid harsh soaps and detergents and maybe, if it’s really dry, get a humidifier. It’s always better to prevent something than to treat it.”

MORE TIPS FROM DERMATOLOGISTS

Want more tips like these? NBC News BETTER is obsessed with finding easier, healthier and smarter ways to live. Sign up for our newsletter and follow us on Facebook, Twitter and Instagram.

Источник: https://www.nbcnews.com/select/lifestyle/how-care-dry-cracked-heels-according-dermatologists-ncna1080001

20 Flame Test Lab. Use dilute or approximately 0. Give the product for the following reactions. We have tutors online 24/7 who can help you get unstuck. Bookmark File PDF Flame Test Lab 10 Answer Key Flame Test Lab Activity Key flame test. Burn the loop end of the wire to remove any dust. This test works well for metal ions, and was perfected by Robert Bunsen (1811 – 1899). Use flame tests to identify a metal or metallic salt by the color that it produces when it is put into a flame. Ask Expert Tutors You can ask You can ask You can ask (will expire ). Metal/Flame Color λ (nm) λ (m) ΔE (J) Post-Lab Questions. Metal Color of Flame Data Table 2 Metal/Color of Flame λ (nm) λ (m) ΔE (J) Post-Lab Questions 1. In this unit we are learning to identify trends and properties of atoms, and in doing so, we will look at the colors these elements give off based. nova official website search for the super battery. Procedure: 1. in China) until the 1830s, all fireworks were either white or orange. Learn how in 5 minutes with a tutorial resource. Lab 6, Experiment 2: Flame Tests: Using the color of a salt in a flame to identify certain metal ions. *If you were absent for either of the replacement reaction labs, download the directions and complete the lab by following the directions but looking up the different reactions on youtube to use as your observations. When placed in a flame, aluminum does not change the flame's color, and so a visual flame test cannot be used to show the presence of Al. 2 Move any flammable materials away from work area 3 Attach the connecting hose. In this lab you will learn how to light and adjust a burner flame and to locate the hottest part of the flame. Pre-laboratory Assignment: Flame Tests of Metal Cations. This test works well for metal ions, and was perfected by Robert Bunsen (1811 – 1899). In the flame test if this energy has the form of visible light theflamewill produce a color characteristic of the element. Results Table. 3 years ago by. LAB: PROPERTIES OF IONIC COMPOUNDS (50pts) Introduction. To do a flame test on a metallic element, the metal is first dissolved in a solution and the solution is then held in the hot, blue flame of a Bunsen burner. Flame test tubes in the older symbols answer key is a question if your book online. [VIEW] Flame Test 1. Position the test tube so that it contacts the blue inner core of the flame. The students then use this information to identify two unknown salts. Worksheets may also be considered as the application form of the question bank principle to teach scholar intelligence. Flame Test Lab. Although the data entered in the sheets are to help one out, it can be a puzzle for one’s mind as well. An exothermic process releases heat, causing the temperature of the immediate surroundings to rise. Flame Test Lab Activity Key Note: If chloride compounds are not available, metal nitrate compounds may be substituted. Lab 13: Urinary Systempage 54 Lab 14: Digestive System and Acid/Base Demopage 60 At the end of each lab, you should be able to understand the concepts and processes learned and answer any of the questions investigated in the activity. Acids, Bases, and Salts Video Post Test. We have tutors online 24/7 who can help you get unstuck. When the electron "falls" back to its ground state, energy is given off in the form of light. Add 15 drops of each 0. Method 2: Take the nichrome or platinum wire and create a small loop at the end by bending the wire. Bookmark File PDF Flame Test Lab 10 Answer Key Flame Test Lab Activity Key flame test. The Flame Test lab was done in several parts. Use Table 1 in the. Pre-Lab Assignment Before coming to lab: • Read the lab thoroughly. Convert each of the wavelengths in the Data Table from nanometers to meters. Do NOT add rows to the data tables. Most people probably remember doing this experiment in school chemistry lessons, if not with the full range of ions shown here, but for the uninitiated a brief. We will be partaking in the flame test lab at a safe distance from the flames – behind your computer screens! Please watch the video below. To do a flame test on a metallic element, the metal is first dissolved in a solution and the solution is then held in the hot, blue flame of a Bunsen burner. Question #258458 from lei. Your work can be viewed below, but no changes can be made. Kind of a lab safety answer key is the older symbols, always add small amounts of information would be found worksheet will fan the words. Some metals don't have a color when they are in a compound (in other words, the compound is white), but if subjected to a flame, they often will emit a color that helps to identify them. Grasp one presoaked toothpick with forceps. 2 Move any flammable materials away from work area 3 Attach the connecting hose. Convert each of the wavelengths in the Data Table from nanometers to meters. courses of study iit gandhinagar. Titration Lab. com Art perfect flame gas grill from ash to flame women rising soul of flame imdalind 4 flame test lab answer key flame test lab worksheet flame wood stove manual autodesk flame user guide country flame wood stove country flame pellet stove flame of recca episode guide char broil flame tamer. Bookmark File PDF Flame Test Lab 10 Answer Key Flame Test Lab Activity Key flame test. The flame test is a procedure used in Chemistry to detect the presence of certain elements based on the element's characteristics The test shows the identity of the substance being tested. 6 Comments. analytical calibration curve from flame test lab worksheet answer key , source:terpconnect. You should perform the test a few times for each solution. Be sure to show all work, round answers, and include units on all answers. Flame Test Lab Metal Ions Answer Key emission spectroscopy element identification. You can read Flame Test Lab Answers PDF direct on your mobile phones or PC. Metal/Flame Color λ (nm) λ (m) ΔE (J) Post-Lab Questions. The physical properties of a substance such as flame color, crystal structure, solubility, conductivity and melting point of a substance tell us a lot about the type of bonding in a compound. Most people probably remember doing this experiment in school chemistry lessons, if not with the full range of ions shown here, but for the uninitiated a brief explanation of the origin of the colours follows. pdf FREE PDF DOWNLOAD The energy levels in atoms and ions are the key to the production and detection of light. Periodic Properties Lab with lab write-up; Light waves introduction lesson with notes. Is the flame color a test for the metal or for the chloride in each compound? Explain your answer? 2. This set covers a unit on cell structure and physiology. Defend your answer. Silver nitrate is added to the solution to give a pale creamy colored precipitate. 1) Does your reaction have oxygen as one of it's reactants • In a flame test, copper ions emit a deep blue-green light, much more blue than the flame test for barium. To do a flame test on a metallic element, the metal is first dissolved in a solution and the solution is then held in the hot, blue flame of a Bunsen burner. Flame Test 1. This test works well for metal ions, and was perfected by Robert Bunsen (1811 – 1899). This lab is provided as a hands on way to test an unknown for the identity of cation and anion by using a flame; Question: Module 5 Lab Report - CHEM 1806 Anions, Cations, and Ionic Reactions Follow the directions in the lab write up. Flame Test And Spectroscopy Lab Report flame test part 1 pre lab ucsb mrsec. Ask Expert Tutors You can ask You can ask You can ask (will expire ). After calculating and then preparing specific molarity solutions of strontium chloride, copper II chloride and potassium chloride (good practice!), students observe the distinct colors. Periodic Properties Lab with lab write-up; Light waves introduction lesson with notes. Start studying Flame Test Pre-Lab Quiz. Hold the test tube at a slight angle over the candle flame. Metal Ion Color of Flame Calcium. 5 flame tests and atomic spectra experiment. Using a test tube holder, hold the test tube (at a 45° angle pointed away from people) over the Bunsen burner flame for 3 minutes or until completely black. Note for Teachers : Showing the NOVA film about fireworks (titled Fireworks!) before or after this lab activity may enhance the learning it is meant to produce. see results table above. In the flame test if this energy has the form of visible light theflamewill produce a color characteristic of the element. An unknown solution gives a brick-red flame test. pdf - Flame Test Lab Questions Answer Key 1 Flame Test Lab. susanazamorano. The secondary purpose of the lab was to identify unknown compounds that we would test and then guess as to what they were. *If you were absent for either of the replacement reaction labs, download the directions and complete the lab by following the directions but looking up the different reactions on youtube to use as your observations. docx Answer Key. Materials: Spark lighter Bunsen Burner support stand w/ ring wire screen gloves 250 mL beaker 100mL graduated cylinder metal thermometer Part 1: Parts of a Bunsen Burner! 1. Write the electron configuration of iron and the iron(III) or ferric ion. Basically, this means trying to find patterns of the flame color when. Lab Electron Configuration of Atoms and Ions Honors Chemistry from flame test lab worksheet answer key , source:yumpu. 5 flame tests and atomic spectra experiment. Dip the loop into the lithium salt solution. Record the wavelengths in meters in the. Introduction: When energy is added to an electron, it "jumps " from its ground state to a higher energy level. Silver nitrate is added to the solution to give a pale creamy colored precipitate. Procedure: For this lab we got a whole bunch of compounds and burned them to observe the flame and frequency based off the color of the flame. What is released when an electron loses energy? 3. A fuel-rich (cool) flame is yellow and sooty. This lab is provided as a hands on way to test an unknown for the identity of cation and anion by using a flame; Question: Module 5 Lab Report - CHEM 1806 Anions, Cations, and Ionic Reactions Follow the directions in the lab write up. Finally, observe the various colors that will appear based on the element that is tested. Flame Test And Spectroscopy Lab Report flame test part 1 pre lab ucsb mrsec. Electron energy and light lab answer key. 4 11/20/15 Pre-Lab Questions. This graphic looks at the colour of various metal and metalloid ions that occur during flame tests. Experiment Purpose : 1. No two metals emit the exact same color. Zubrick Class pak 1 two-hour final exam (150 points) Three Exams (67% of final grade). Metal Color of Flame Data Table 2 Metal/Color of Flame λ (nm) λ (m) ΔE (J) Post-Lab Questions 1. flame test wikipedia. End of The Experiment : October 18th, 2014 at 10. Then, in the 1830s, the Italians discovered that adding metal salts to the fireworks mixture resulted in interesting colors, just like in the flame test in this science activity. Chemists began studying colored flames in the 18th century and soon used "flame tests" to distinguish between some elements. Answers in as fast as 15 minutes. Flame test post lab answer key - sjyp. During emission, an electron goes from the excited to the ground state. Hold the splint in a low bunsen burner flame and record the main color that is emitted (other than the color of the flame) in. It was difficult to identify a couple of the elements that had colors that were similar. Method 2: Take the nichrome or platinum wire and create a small loop at the end by bending the wire. Flame Test Lab Worksheet Answer Key with 22 Best Chemistry Unit 4 Review Images On Pinterest. This experiment is a classic, must-do experiment in your chemistry class! dichotomous key (6) differentiation (1) diffusion (1) digestive system (1) digital resources (14) disney (1) distance learning (15). This test works well for metal ions, and was perfected by Robert Bunsen (1811 – 1899). Complete the: Flame test virtual lab; Answer the Pre and Post lab questions: Flame test with lab write-up; Complete a Lab write-up; Electron configuration notes. Give the product for the following reactions. After watching the video, please answer the questions below. Adjust the burner tube to produce a hot flame. This test works well for metal ions, and was perfected by Robert Bunsen (1811 – 1899). Experiment Purpose : 1. When placed in a flame, aluminum does not change the flame's color, and so a visual flame test cannot be used to show the presence of Al. Pre Lab Flame Tests Lab in your lab notebook Finish Calculations for light properties worksheet for homework if you did not complete it in class. The Flame Test lab was done in several parts. Each word document, safety answer key is for the gaps using the gaps only, scientific method name science lab safety symbols. Important! Before you view the answer key, decide whether or not you plan to request an extension. See Figures 3 and 4. Perform a flame test on an unknown metal chloride and record its characteristic color(s) and the probable identity of the unknown in the Data Table. Introduction to the Flame Test Lab: The Flame Test lab was an in-class lab where we tested chemicals in the flames to see the wide range of colors in the color spectrum. Hold the splint in the flame and record the color of the flame that is produced. Most people probably remember doing this experiment in school chemistry lessons, if not with the full range of ions shown here, but for the uninitiated a brief explanation of the origin of the colours follows. Why do different metals have different characteristic flame test colors? 3. Add 15 drops of each 0. Dip the loop into the lithium salt solution. conventional weapons atomic rockets projectrho com. We have tutors online 24/7 who can help you get unstuck. Bookmark File PDF Flame Test Lab 10 Answer Key Flame Test Lab Activity Key flame test. In addition to this, a printable 22 page activity booklet contains student activities. The flame test is a procedure used in Chemistry to detect the presence of certain elements based on the element's characteristics. docx Answer Key. The estimating worksheet is designed to direct. Found 6257 results for: Flame Test Lab Worksheet Answer Key [DOWNLOAD] Flame Test Lab Worksheet Answer Key. 4 11/20/15 Pre-Lab Questions. Flame Test Lab Pre-AP Mr. Get the app ». So I need two more, I considered the fact that a lot of times the. Although the data entered in the sheets are to help one out, it can be a puzzle for one's mind as well. Atwood Hydro Flame Furnace - Scholarships. 2 Move any flammable materials away from work area 3 Attach the connecting hose. Post lab questions page 114 - 115. Electron energy and light lab answer key. senior chemistry extended experimental investigations. Flame Test Post Lab Quiz Flashcards

20 Flame Test Lab. Use dilute or approximately 0. Give the product for the following reactions. We have tutors online 24/7 who can help you get unstuck. Bookmark File PDF Flame Test Lab 10 Answer Key Flame Test Lab Activity Key flame test. Burn the loop end of the wire to remove any dust. This test works well for metal ions, and was perfected by Robert Bunsen (1811 – 1899). Use flame tests to identify a metal or metallic salt by the color that it produces when it is put into a flame. Ask Expert Tutors You can ask You can ask You can ask (will expire ). Metal/Flame Color λ (nm) λ (m) ΔE (J) Post-Lab Questions. Metal Color of Flame Data Table 2 Metal/Color of Flame λ (nm) λ (m) ΔE (J) Post-Lab Questions 1. In this unit we are learning to identify trends and properties of atoms, and in doing so, we will look at the colors these elements give off based. nova official website search for the super battery. Procedure: 1. in China) until the 1830s, all fireworks were either white or orange. Learn how in 5 minutes with a tutorial resource. Lab 6, Experiment 2: Flame Tests: Using the color of a salt in a flame to identify certain metal ions. *If you were absent for either of the replacement reaction labs, download the directions and complete the lab by following the directions but looking up the different reactions on youtube to use as your observations. When placed in a flame, aluminum does not change the flame's color, and so a visual flame test cannot be used to show the presence of Al. 2 Move any flammable materials away from work area 3 Attach the connecting hose. In this lab you will learn how to light and adjust a burner flame and to locate the hottest part of the flame. Pre-laboratory Assignment: Flame Tests of Metal Cations. This test works well for metal ions, and was perfected by Robert Bunsen (1811 – 1899). In the flame test if this energy has the form of visible light theflamewill produce a color characteristic of the element. Results Table. 3 years ago by. LAB: PROPERTIES OF IONIC COMPOUNDS (50pts) Introduction. To do a flame test on a metallic element, the metal is first dissolved in a solution and the solution is then held in the hot, blue flame of a Bunsen burner. Flame test tubes in the older symbols answer key is a question if your book online. [VIEW] Flame Test 1. Position the test tube so that it contacts the blue inner core of the flame. The students then use this information to identify two USB Disk Security 6.7.0.0 Crcak product key - Crack Key For U salts. Worksheets may also be considered as the application form of the question bank principle to teach scholar intelligence. Flame Test Lab. Although the data entered in the sheets are to help one out, it calibre drm removal be a puzzle for one’s mind as well. An exothermic process releases heat, causing the temperature of the immediate surroundings to rise. Flame Test Lab Activity Key Note: If chloride compounds are not available, metal nitrate compounds may be substituted. Lab 13: Urinary Systempage 54 Lab 14: Digestive System and Acid/Base Demopage 60 At the end of each lab, you should be able to understand the concepts and processes learned and answer any of the questions investigated in the activity. Acids, Bases, and Salts Video Post Test. We have tutors online 24/7 who can help you get unstuck. When the electron "falls" back to its ground state, energy is given off in the form of light. Add 15 drops of each 0. Method 2: Take the nichrome or platinum wire and create a small loop at the end by bending the wire. Bookmark File PDF Flame Test Lab 10 Answer Key Flame Test Lab Activity Key flame test. The Flame Test lab was done in several parts. Use Table 1 in the. Pre-Lab Assignment Before coming to lab: • Read the lab thoroughly. Convert each of the wavelengths in the Data Table from nanometers to meters. Do NOT add rows to the data tables. Most people probably remember doing this experiment in school chemistry lessons, if not with the full range of ions shown here, smadav 2018 registration key - Free Activators for the uninitiated a brief. We will be partaking in the flame test lab at a safe distance from the flames – behind your computer screens! Please watch the video below. To do a flame test on a metallic element, the metal is first dissolved in a solution and the solution is then held in the hot, blue flame of a Bunsen burner. Question #258458 from lei. Your work can be viewed below, but no changes can be made. Kind of a lab safety answer key is the older symbols, always add small amounts of information would be found worksheet will fan the words. Some metals don't have a color when they are in a compound (in other words, the compound is white), but if subjected to a flame, they often will emit a color that helps to identify them. Grasp one presoaked toothpick with forceps. 2 Move any flammable materials away from work area 3 Attach the connecting hose. Convert each of the wavelengths in the Data Table from nanometers to meters. courses of study iit gandhinagar. Titration Lab. com Art perfect flame gas grill from ash to flame women rising soul of flame imdalind 4 flame test lab answer key flame test lab worksheet flame wood stove manual autodesk flame user guide country flame wood stove country flame pellet stove flame of recca episode guide char broil flame tamer. Bookmark File PDF Flame Test Lab 10 Answer Key Flame Test Lab Activity Key flame test. The flame test is a procedure used in Chemistry to detect the presence of certain elements based on the element's characteristics The test shows the identity of the substance being tested. 6 Comments. analytical calibration curve from flame test lab worksheet answer keysource:terpconnect. You should perform the test a few times for each solution. Be sure to show all work, round answers, and include units on all answers. Flame Test Lab Metal Ions Answer Key emission spectroscopy element identification. You can read Flame Test Lab Answers PDF direct on your mobile phones or PC. Metal/Flame Color λ (nm) λ (m) ΔE (J) Post-Lab Questions. The physical properties of a substance such as flame color, crystal structure, solubility, conductivity and melting point of a substance tell us a lot about the type of bonding in a compound. Most people probably remember doing this experiment in school chemistry lessons, if not with the full range of ions shown here, but for the uninitiated a brief explanation of the origin of the colours follows. pdf FREE PDF DOWNLOAD The energy levels in atoms and ions are the key to the production and detection of light. Periodic Properties Lab with lab write-up; Light waves introduction lesson with notes. Is the flame color a test for the metal or for the chloride in each compound? Explain your answer? 2. This set covers a unit on cell structure and physiology. Defend your answer. Silver nitrate is added to the solution to give a pale creamy colored precipitate. 1) Does your reaction have oxygen as one of it's reactants • In a flame test, copper ions emit a deep blue-green light, much more blue than the flame test for barium. To do a flame test on a metallic element, the metal is first dissolved in a solution and the solution is then held in the hot, blue flame of a Bunsen burner. Flame Test 1. This test works well for metal ions, and was perfected by Robert Bunsen (1811 – 1899). This lab is provided as a hands on way to test an unknown for the identity of cation and anion by using a flame; Question: Module 5 Lab Report - CHEM 1806 Anions, Cations, and Ionic Reactions Follow the directions in the lab write up. Flame Test And Spectroscopy Lab Report flame test part 1 pre lab ucsb mrsec. Ask Expert Tutors You can ask You can ask You can ask (will expire ). After calculating and then preparing specific molarity solutions of strontium chloride, copper II chloride and potassium chloride (good practice!), students observe the distinct colors. Periodic Properties Lab with lab write-up; Light waves introduction lesson with notes. Start studying Flame Test Pre-Lab Quiz. Hold the test tube at a slight angle over the candle flame. Metal Ion Color of Flame Calcium. 5 flame tests and atomic spectra experiment. Using a test tube holder, hold the test tube (at a 45° angle pointed away from people) over the Bunsen burner flame for 3 minutes or until Reason 12.0.0 Crack + Activation Code Free Download black. Note for Teachers : Showing the NOVA film about fireworks (titled Fireworks!) before or after this lab activity may enhance the learning it is meant to produce. see results table above. In the flame test if this energy has the form of visible light theflamewill produce a color characteristic of the element. An unknown solution gives a brick-red flame test. pdf - Flame Test Lab Questions Answer Key 1 Flame Test Lab. susanazamorano. The secondary purpose of the lab was to identify unknown compounds that we would test and then guess as to what they were. *If you were absent for either of the replacement reaction labs, download the directions and complete the lab by following the directions but looking up the different reactions on youtube to use as your observations. docx Answer Key. Materials: Spark lighter Bunsen Burner support stand w/ ring wire screen gloves 250 mL beaker 100mL graduated cylinder metal thermometer Part 1: Parts of a Bunsen Burner! 1. Write the electron configuration of iron and the iron(III) or ferric ion. Basically, this means trying to find patterns of the flame color when. Lab Electron Configuration of Atoms and Ions Honors Chemistry from flame test lab worksheet answer keysource:yumpu. 5 flame tests and atomic spectra experiment. Dip the loop into the lithium salt solution. Record the wavelengths in meters in the. Introduction: When energy is added to an electron, it "jumps " VMware Tools 15 Free Download and How to Install its ground state to a higher energy level. Silver nitrate is added to the solution to give a pale creamy colored precipitate. Procedure: For this lab we got a whole bunch of compounds and burned them to observe the flame and frequency based off the color of the flame. What is released when an electron loses energy? 3. A fuel-rich (cool) flame is yellow and sooty. This lab is provided as a hands on way vegas movie studio test an unknown for the identity of cation and anion by using a flame; Question: Module 5 Lab Report - CHEM 1806 Anions, Cations, and Ionic Reactions Follow the directions in the lab write up. Finally, observe the various colors that will appear based on the element that is tested. Flame Test Advanced system repair pro 2019 - Crack Key For U Spectroscopy Lab Report flame test part 1 pre lab ucsb mrsec. Electron energy and light lab answer key. 4 11/20/15 Pre-Lab Questions. This graphic looks at the colour of various metal and metalloid ions that occur during flame tests. Experiment Purpose : 1. No two metals emit the exact same color. Zubrick Class pak 1 two-hour final exam (150 points) Three Exams (67% of final grade). Metal Color of Flame Data Table 2 Metal/Color of Flame λ (nm) λ (m) ΔE (J) Post-Lab Questions 1. flame test wikipedia. End of The Experiment : October 18th, 2014 at 10. Then, in the 1830s, the Italians discovered that adding metal salts to the fireworks mixture resulted in interesting colors, just like in the flame test in this science activity. Chemists began studying colored flames in the 18th century and soon used "flame tests" to distinguish between some elements. Answers in as fast as 15 minutes. Flame test post lab answer key - sjyp. During emission, an electron goes from the excited to the ground state. Hold the splint in a low bunsen burner flame and record the main color that is emitted (other than the color of the flame) in. It was difficult to identify a couple of the elements that had colors that were similar. Method 2: Take the nichrome or platinum wire and create a small loop at the end by bending the wire. Flame Test Lab Worksheet Answer Key with 22 Best Chemistry Unit 4 Review Images On Pinterest. This experiment is a classic, must-do experiment in your chemistry class! dichotomous key (6) differentiation (1) diffusion (1) digestive system (1) digital resources (14) disney (1) distance learning (15). This test works well for metal ions, and was perfected by Robert Bunsen (1811 – 1899). Complete the: Flame test virtual lab; Answer the Pre and Post lab questions: Flame test with lab write-up; Complete a Lab write-up; Electron configuration notes. Give the product for the following reactions. After watching the video, please answer the questions below. Adjust the burner tube to produce a hot flame. This test works well for metal ions, and was perfected by Robert Bunsen (1811 – 1899). Experiment Purpose : 1. When placed in a flame, aluminum does not change the flame's color, and so a visual flame test cannot be used to show the presence of Al. Pre Lab Flame Tests Lab in your lab notebook Finish Calculations for light properties worksheet for homework if you did not complete it in class. The Flame Test lab was done in several parts. Each word document, safety answer key is for acid properties - Crack Key For U gaps using the gaps only, scientific method name science lab safety symbols. Important! Before you view the answer key, decide whether or not you plan to request an extension. See Figures 3 and 4. Perform a flame test on an unknown metal chloride and record its characteristic color(s) and the probable identity of the unknown in the Data Table. Introduction to the Flame Test Lab: The Flame Test lab was an in-class lab where we tested chemicals in the flames to see the wide range of colors in the color spectrum. Hold the splint in the flame and record the color of the flame that is produced. Most people probably remember doing this experiment in school chemistry lessons, if not with the full range of ions shown here, but for the uninitiated a brief explanation of the origin of the colours follows. Why do different metals have different characteristic flame test colors? 3. Add 15 drops of each 0. Dip the loop into the lithium salt solution. conventional weapons atomic rockets projectrho com. We have tutors online 24/7 who can help you get unstuck. Bookmark File PDF Flame Test Lab 10 Answer Key Flame Test Lab Activity Key flame test. In addition to this, a printable 22 page activity booklet contains student activities. The flame test is a procedure used in Chemistry to detect the presence of certain elements based on the element's characteristics. docx Answer Key. The estimating worksheet is designed to direct. Found 6257 results for: Flame Test Lab Worksheet Answer Key [DOWNLOAD] Flame Test Lab Worksheet Answer Key. 4 11/20/15 Pre-Lab Questions. Flame Test Lab Pre-AP Mr. Get the app ». So I need two more, I considered the fact that a lot of times the. Although the data entered in the sheets are to help one out, it can be a puzzle for one's mind as well. Atwood Hydro Flame Furnace - Scholarships. 2 Move any flammable materials away from work area 3 Attach the connecting hose. Post lab questions page 114 - 115. Electron energy and light lab answer key. senior chemistry extended experimental investigations. Flame Test Post Lab Quiz Flashcards

Acid Rain

Inorganic Reactions Experiment

Authors: Rachel Casiday and Regina Frey
Department of Chemistry, Washington University
St. Louis, MO 63130


Natural Acidity of Rainwater

Pure water has a pH of 7.0 (neutral); however, natural, unpolluted rainwater actually has a pH of about 5.6 (acidic).[Recall from Experiment 1 that pH is a measure of the hydrogen ion (H+) concentration.] The acidity of rainwater comes from the natural presence of three substances (CO2, NO, and SO2) found in the troposphere (the lowest layer of the atmosphere). As is seen in Table I, carbon dioxide (CO2) is present in the greatest concentration and therefore contributes the most to the natural acidity of rainwater.

Gas

Natural Sources

Concentration

Carbon dioxide
CO2
Decomposition 355 ppm
Nitric oxide
NO
Electric discharge 0.01 ppm
Sulfur dioxide
SO2
Volcanic gases 0-0.01 ppm

Table 1

Carbon dioxide, produced in the decomposition of organic material, is the primary source of acidity in unpolluted rainwater.

NOTE: Parts per million (ppm) is a common concentration measure used in environmental chemistry. The formula for ppm is given by:

Carbon dioxide reacts with water to form carbonic acid (Equation 1). Carbonic acid then dissociates to give the hydrogen ion (H+) and the hydrogen carbonate ion (HCO3-) (Equation 2). The ability of H2CO3 to deliver H+ is what classifies this molecule as an acid, thus lowering the pH of a solution.


(1)

 


(2)

Nitric oxide (NO), which also contributes to the natural acidity of rainwater, is formed during lightning storms by the reaction of nitrogen and oxygen, two common atmospheric gases (Equation 3). In air, NO is oxidized to nitrogen dioxide (NO2) (Equation 4), which in turn reacts with water to give nitric acid (HNO3) (Equation 5). This acid dissociates in water to yield hydrogen ions and nitrate ions (NO3-) in a reaction analagous to the dissociation of carbonic acid shown in Equation 2, again lowering the pH of the solution.


(3)

 


(4)

 


(5)

Acidity of Polluted Rainwater

Unfortunately, human industrial activity produces additional acid-forming compounds in far greater quantities than the natural sources of acidity described above. In some areas of the United States, the pH of rainwater can be 3.0 or lower, approximately 1000 times more acidic than normal rainwater. In 1982, the pH of a fog on the West Coast of the United States was measured at 1.8! When rainwater is too acidic, it can cause problems ranging from killing freshwater fish and damaging crops, to eroding buildings and monuments.


Questions on Acidity of Rainwater

1. List two or more ways that you could test the acidity of a sample of rainwater.

2. Write a balanced chemical equation for the dissociation of nitric acid in water. (HINT: Draw an analogy with Equation 2.)

3. The gaseous oxides found in the atmosphere, including CO2 and NO are nonmetal oxides. What would happen to the pH of rainwater if the atmosphere contained metal oxides instead? (HINT: Think back to Experiment 1.) Briefly, explain your answer.


Sources of Excess Acidity in Rainwater

What causes such a dramatic increase in the acidity of rain relative to pure water? The answer lies within the concentrations of nitric oxide and sulfur dioxide in polluted air. As shown in Table II and Figure 1, the concentrations of these oxides are much higher than in clean air.

Gas

Non-Natural Sources

Concentration

Nitric oxide
NO
Internal Combustion0.2 ppm
Sulfur dioxide
SO2
Fossil-fuel Combustion 0.1 - 2.0 ppm

Table II

Humans cause many combustion processes that dramatically increase the concentrations of acid-producing oxides in the atmosphere. Although CO2 is present in a much higher concentration than NO and SO2, CO2 does not form acid to the same extent as the other two gases. Thus, a large increase in the concentration of NO and SO2 significantly affects the pH of rainwater, even though both gases are present at much lower concentration than CO2.

Figure 1

Comparison of the concentrations of NO and SO2 in clean and polluted air.

About one-fourth of the acidity of rain is accounted for by nitric acid (HNO3). In addition to the natural processes that form small amounts of nitric acid in rainwater, high-temperature air combustion, such as occurs in car engines and power plants, produces large amounts of NO gas. This gas then forms nitric acid via Equations 4 and 5. Thus, a process that occurs naturally at levels tolerable by the environment can harm the environment when human activity causes the process (e.g., formation of nitric acid) to occur to a much greater extent.

What about the other 75% of the acidity of rain? Most is accounted for by the presence of sulfuric acid (H2SO4) in rainwater. Although sulfuric acid may be produced naturally in small quantities from biological decay and volcanic activity (Figure 1), it is produced almost entirely by human activity, especially the combustion of sulfur-containing fossil fuels in power plants. When these fossil fuels are burned, the sulfur contained in them reacts with oxygen from the air to form sulfur dioxide (SO2). Combustion of fossil fuels accounts for approximately 80% of the total atmospheric SO2 in the United States. The effects of burning fossil fuels can be dramatic: in contrast to the unpolluted atmospheric SO2 concentration of 0 to 0.01 ppm, polluted urban air can contain 0.1 to 2 ppm SO2, or up to 200 times more SO2! Sulfur dioxide, like the oxides of carbon and nitrogen, reacts with water to form sulfuric acid (Equation 6).


(6)

Sulfuric acid is a strong acid, so it readily dissociates in water, to give an H+ ion and an HSO4- ion (Equation 7). The HSO4- ion may further dissociate to give H+ and SO42- (Equation 8). Thus, the presence of H2SO4 causes the concentration of H+ ions to increase dramatically, and so the pH of the rainwater drops to harmful levels.


(7)

 


(8)


Questions on Sources of Acidity in Rainwater

4. At sea level and 25oC, one mole of air fills a volume of 24.5 liters, and the density of air is 1.22x10-6 g/ml. Compute the mole fraction (i.e.,moles of component /total moles) and molarity of SO2 when the atmospheric concentration of SO2 is 2.0 ppm (see note in Table I).

5.One strategy for limiting the amount of acid pollution in the atmosphere is scrubbing. In particular, calcium oxide (CaO) is injected into the combustion chamber of a power plant, where it reacts with the sulfur dioxide produced, to yield solid calcium sulfite.

a. Write a balanced chemical equation for this reaction. (HINT: Consult the table of common ions in the tutorial assignment for Experiment 1 to view the structure and formula for sulfite; also, use your knowledge of the periodic table to deduce the charge of the calcium ion. Using these facts, you can deduce the formula for calcium sulfite.)

b. Approximately one ton, or 9.0x102 kg, of calcium sulfite is generated each year for every person served by a power plant. How much sulfur dioxide (in moles) is prevented from entering the atmosphere when this much calcium sulfite is generated? Show your calculation.

c. The final stage in the scrubbing process is to treat the combustion gases with a slurry of solid CaO in water, in order to trap any remaining SO2 and convert it to calcium sulfite. A slurry is a thick suspension of an insoluble precipitate in water. Using the solubility guidelines provided in the lab manual for this experiment, predict whether this stage of the scrubbing process will produce a slurry (i.e., precipitate) or a solution (i.e., no precipitate) of calcium sulfite.

d. If MgO, rather than CaO, were used for scrubbing, would the product of the final stage be a slurry or a solution of magnesium sulfite? (Assume that a very large quantity of grammarly app for mac - Crack Key For U magnesium sulfite, relative to the amount of water, is produced.)


Environmental Effects of Acid Rain

Acid rain triggers a number of inorganic and biochemical reactions with deleterious environmental effects, making this a growing environmental problem worldwide.

  • Many lakes have become so acidic that fish cannot live in them anymore.
  • Degradation of many soil minerals produces metal ions that are then washed away in the runoff, causing several effects:
  • Atmospheric pollutants are easily moved by wind currents, so acid-rain effects are felt far from where pollutants are generated.

Stone Buildings and Monuments in Acid Rain

Marble and limestone have long been preferred materials for constructing durable buildings and monuments. The Saint Louis Art Museum, the Parthenon in Greece, the Chicago Field Museum, and the United States Capitol building are all made of these materials. Marble and limestone both consist of calcium carbonate (CaCO3), and differ only in their crystalline structure. Limestone consists of smaller crystals and is more porous than marble; it is used more extensively in buildings. Marble, with its larger crystals and smaller pores, can attain a high polish and is thus preferred for monuments and statues. Although these are recognized as highly durable materials, buildings and outdoor monuments made of marble and limestone are now being gradually eroded away by acid rain.

How does this happen? A chemical reaction (Equation 9) between calcium carbonate and sulfuric acid (the primary acid component of acid rain) results in the dissolution of CaCO3 to give aqueous ions, which in turn are washed away in the water flow.


(9)

This process occurs at the surface of the buildings or monuments; thus acid rain can easily destroy the details on relief work (e.g., the faces on a statue), but generally does not affect the structural integrity of the building. The degree of damage is determined not only by the acidity of the rainwater, but also by the amount of water flow that a region of the surface receives. Regions exposed to direct downpour of acid rain are highly susceptible to erosion, but regions that are more sheltered from water flow (such as under eaves and overhangs of limestone buildings) are much better preserved. The marble columns of the emperors Marcus Aurelius and Trajan, in Rome, provide a striking example: large volumes of rainwater flow directly over certain parts of the columns, which have been badly eroded; other parts are protected by wind effects from this flow, and are in extremely good condition even after nearly 2000 years!

Even those parts of marble and limestone structures that are not themselves eroded can be damaged by this process (Equation 9). When the water dries, it leaves behind the ions that were dissolved in it. When a solution containing calcium and sulfate ions dries, the ions crystallize as CaSO4l 2H2O, which is gypsum. Gypsum is soluble in water, so it is washed away from areas that receive a heavy flow of rain. However, gypsum accumulates in the same sheltered areas that are protected from erosion, and attracts dust, carbon particles, dry-ash, and other dark pollutants. This results in blackening of the surfaces where gypsum accumulates.

An even more serious situation arises when water containing calcium and sulfate ions penetrates the stone's pores. When the water dries, the ions form salt crystals within the pore system. These crystals can disrupt the crystalline arrangement of the atoms in the stone, causing the fundamental structure of the stone to be disturbed. If the crystalline structure is disrupted sufficiently, the stone may actually crack. Thus, porosity is an important factor in determining a stone's durability.


Questions on Effects of Acid Rain

6. Based on the information described above about the calcium ion, and the formula of calcium carbonate (CaCO3), deduce the charge of the carbonate ion. Also, in the structure of the carbonate ion, are any of the oxygens bonded to one another, or all the oxygens bonded to the carbon atom? (HINT: Consult the structure of the common ions given in the tutorial for Experiment 1).

7. In water, H2SO4 can dissociate to yield two H+ ions and one SO42- ion. Write the net ionic equation for the reaction of calcium carbonate and sulfuric acid. (See the introduction to Experiment 2 in the lab manual for a discussion of net ionic equations.)

8. Which is a more durable building material, limestone or marble? Briefly, explain your reasoning.

Additional Links:


References:

Brown, Lemay, and Buster. Chemistry: the Central Science, 7th ed. Upper Saddle River, NJ: Prentice Hall, 1997. p. 673-5.

Charola, A. "Acid Rain Effects on Stone Monuments," J. Chem. Ed.64 (1987), p. 436-7.

Petrucci and Harwood. General Chemistry: Principles and Modern Applications, 7th ed. Upper Saddle River, NJ: Prentice Hall, 1997. p. 614-5.

Walk, M. F. and P.J. Godfrey. "Effects of Acid Deposition on Surface Waters," J. New England Water Works Assn. Dec. 1990, p. 248-251.

Zumdahl, S. Chem. Principles, 3rd ed. Boston: Houghton Mifflin, 1998. p. 174-6.

Stryer, L. Biochemistry, 4th ed., W.H. Freeman and Co., New York, 1995, p. 332-339.


Acknowledgements:

The authors thank Dewey Holten (Washington University) for many helpful suggestions in the writing of this tutorial.

The development of this tutorial was supported by a grant from the Howard Hughes Medical Institute, through the Undergraduate Biological Sciences Education program, Grant HHMI# 71192-502004 to Washington University.

Copyright 1998, Washington University, All Rights Reserved.

Источник: http://www.chemistry.wustl.edu/~edudev/LabTutorials/Water/FreshWater/acidrain.html

Six-mark questions

Six-mark questions are extended open response questions. These require longer answers than the structured questions. It is wise to plan your answer rather than rushing straight into it, otherwise you may stray away from the key points.

To gain full marks you need to:

  • support explanations using scientific knowledge and understanding
  • use appropriate scientific words and terms
  • write clearly and link ideas in a logical way
  • maintain a sustained line of reasoning, rather than getting lost or bogged down

Six-mark questions often use these command words:

  • describe - you need to give an account but no reason
  • explain - you must give reasons or explanations
  • devise - you must plan or invent a procedure using your scientific knowledge and understanding
  • evaluate - you must review information, including identifying strengths and weaknesses, and make a supported conclusion

Six-mark questions may be synoptic questions. These questions bring together ideas from two or more topics. For example, a question about fertilisers could include ideas about covalent substances, acids and alkalis, chemical calculations and effects on the environment.

The answers shown here give marking points as bullet points. You do not usually need to include all of them to gain six marks but you do need to write in full sentences, linking them logically and clearly.

Edexcel questions courtesy of Pearson Education Ltd.

Sample question 1 - Foundation

Question

Marble chips react with dilute hydrochloric acid to produce carbon dioxide gas.

The rate of this reaction can be changed by changing the size of the marble chips.

Describe how you could investigate what effect using smaller marble chips has on the rate of this reaction. Predict and explain the effect of using smaller marble chips on the reaction rate. [6 marks]

Your answer should include the following:

  • measure volume of acid/stated volume
  • measure mass of marble chips/stated mass
  • add acid to marble or marble to acid in a suitable container, eg flask, beaker, boiling tube, test tube
  • collect the gas in a gas syringe/measuring cylinder over water or bubble gas through limewater/bubble gas through water
  • measure the amount/volume of carbon dioxide or count the bubbles/fixed volume of carbon dioxide
  • measure mass/mass loss (on a balance)
  • time/measure how long the reaction takes
  • repeat experiment with different size marble chips
  • use the same mass of marble chips
  • use the same volume/concentration/mass of acid/same acid
  • crush the marble/use powdered marble

Results:

  • smaller chips (of marble) have a more vigorous
  • reaction/produce more fizzing/bubbles
  • smaller chips take less time to react/produce a certain volume of gas/have a certain mass loss
  • smaller chips have a larger surface area
  • smaller chips react faster
  • larger surface gives a faster reaction

[6]

Sample question 2 - Foundation

Question

Hydrochloric acid reacts with magnesium metal to produce hydrogen gas:

Magnesium + hydrochloric acid → magnesium chloride + hydrogen

Describe how you could use magnesium ribbon, and a solution of hydrochloric acid, to show that decreasing the concentration of the hydrochloric acid changes the rate of this reaction. [6 marks]

Your answer should include the following:

Method:

  • dilute the acid.
  • to make different concentrations
  • add magnesium to acid.
  • in suitable container
  • equal volumes of the acids
  • equal lengths of magnesium

Observations:

  • observe/count bubbles
  • highest concentration magnesium reacts, lowest concentration magnesium does not react
  • time how long it takes for the magnesium to disappear
  • measure volume gas produced
  • measure decrease in mass

Analysis:

  • formed bubbles faster
  • magnesium disappears faster
  • gas produced faster
  • mass lost faster

[6]

Sample question 3 - Higher

Question

A student investigated the rate of reaction between zinc and dilute sulfuric acid:

Zn(s) + H2SO4(aq) → ZnSO4(aq) + H2(g)

The student carried out two experiments, using the same mass of zinc and the same sized pieces of zinc. The results are shown in the table.

Experiment 1Experiment 2
Concentration of sulfuric acid0.5 mol dm-31.5 mol dm-3
Temperature20°C40°C
Rate of reactionSlowFast

Evaluate these results, explaining the reasons why the rate of reaction in Experiment 2 is faster than the rate of reaction in Experiment 1.

In your answer you should refer to the frequency and energy of collisions between particles. [6 marks]

General points:

  • reactions occur when particles collide
  • more frequent collisions cause higher rate of reaction
  • mass and size of zinc pieces stay the same so no effect on rate of reaction
  • because surface area stays the same
  • two factors have been altered in the same experiment
  • cannot be certain of effect of each

Concentration:

  • experiment 2 higher/triple concentration of acid
  • so more particles (in same volume)
  • so more frequent collisions between particles
  • more successful collisions

Temperature:

  • experiment 2 higher temperature
  • particles move faster
  • particles have more energy
  • so more frequent collisions between particles (so increased rate)
  • more successful collisions
  • so more energetic collisions between particles
  • more particles have enough energy to react (activation energy) when they collide

[6]

Sample question 4 - Higher

Question

Reactions can occur when particles collide.

Rates of reactions can be altered by changing conditions.

Explain how the rate of reaction between a solid and a liquid is altered by reducing the size of the pieces of solid and by increasing the temperature of the liquid. [6 marks]

Your answer should include the following:

Smaller pieces of solid:

  • of same mass
  • have larger surface area to volume ratios
  • so more frequent collisions
  • so higher rate of reaction

Higher temperature:

  • particles move faster
  • more frequent collisions
  • particles have more energy
  • more collisions have required energy to react/activation energy
  • more collisions successful
  • higher rate of reaction

[6]

Источник: https://www.bbc.co.uk/bitesize/guides/ztngtv4/revision/6

The dietary supplement industry was valued at $122 billion in 2016, and it continues to grow. [1] One category of dietary supplements are workout supplements, which are typically taken before (‘pre-workout’) or after exercising (‘post-workout’), and are sold in a variety of forms from pills to powders and ready-to-drink shakes. The global pre-workout supplement market size alone was estimated to reach $13.98 billion in 2020 and almost double in size to $23.77 billion by 2027. [2]

Fitness gurus and blogs touting these products as crucial for peak performance, fat loss, and explosive muscle growth in combination with complicated scientific-sounding names and labels might have you believing you can’t effectively exercise without them. But do these supplements live up to the hype, and are they even necessary–or in some cases, safe? Like other dietary supplements, workout supplements are not regulated for safety, so it’s a good idea to research their effects and ingredients and consult with your physician before adding them to your fitness routine.

When we exercise, our bodies use three main sources of fuel: carbohydrates, fat, and protein. Carbohydrates, which are stored in the liver and skeletal muscles in the form of glycogen, provide the most efficient source of energy during exercise. Glycogen can be easily metabolized into glucose, which provides immediate energy to fuel the brain, nervous system, and muscles during exercise. The body’s glycogen supply can provide enough fuel for 90–120 minutes of vigorous activity. The depletion of glycogen stores in the body creates the feeling of “hitting a wall” during exercise. As glycogen stores are depleted, the body begins to break down fat Format Factory 4.6.2.0 Keygen - Crack Key For U burn for fuel, especially during low- to moderate-intensity activity. In the latest stages of prolonged exercise when glycogen stores are at their lowest, the body begins to break down skeletal muscle protein for glucose production. Physical activity can also acid properties - Crack Key For U muscle growth, also known as hypertrophy. Weight lifting and other resistance training exercises are commonly used to increase skeletal muscle mass, but cardiovascular exercise like running can also spur muscle growth. Physical activity causes structural damage to muscle fibers, especially when muscles are challenged with multiple repetitions of heavy weights. The body’s repair response involves fusing broken muscle fibers together to form new muscle protein strands, which in turn increases muscle size. A variety of factors influence how rapidly muscles grow with exercise, including the amount of weight lifted and the number of repetitions.

Learn more about different types of physical activity

Here we review the scientific evidence behind some of the most popular ingredients in workout supplements.

Pre-Workout Supplements

Pre-workout supplements are designed to provide energy and aid endurance throughout a workout. They are typically taken 15-30 minutes before a workout, but can also be consumed during exercise. Below are common ingredients found in pre-workout supplements that the Acid properties - Crack Key For U of Nutrition and Dietetics, Dietitians of Canada, and the American College of Sports Medicine have highlighted as having evidence-based uses in sports nutrition. [3] These supplements have also been categorized as apparently safe and having strong evidence to support efficacy by the International Society of Sports Nutrition. [4] However, it is important to consult a physician or dietitian before acid properties - Crack Key For U these supplements, as they are not regulated for safety. They also may not be necessary for your workout routine, as sports nutrition research and recommendations generally target high-intensity athletes.

Beta-Alanine

Beta-alanine is an amino acid that is produced in the liver and also found in fish, poultry, and meat. When dosed at 4–6g/day for 2–4 weeks, this supplement has been shown to improve exercise performance, particularly for high-intensity exercise lasting 1–4 minutes, such as high-intensity interval training (HIIT) or short sprints. It has also been shown to reduce neuromuscular fatigue, particularly in older adults. [5] How does it work? During exercise the body breaks down glucose into lactic acid, which is then converted into lactate. This produces hydrogen ions, which lower muscle pH levels. This acidity reduces muscles’ ability to contract, causing fatigue. [6] Beta-alanine increases muscle concentrations of carnosine, which is a proton buffer that reduces muscle acidity during high-intensity exercise, which in turn reduces overall fatigue. [5] This supplement is often combined with sodium bicarbonate, or baking soda, which also reduces muscle acidity. A common side effect of beta-alanine supplementation is paresthesia, or a skin tingling sensation, [3] but this effect can be attenuated by taking lower doses (1.6g) or using a sustained-release instead of a rapid-release formula. [5] In short, this supplement can help you exercise at high-intensity for a longer period of time, which could potentially lead to increased muscle mass. The International Society of Sports Nutrition has asserted that “beta-alanine supplementation currently appears to be safe in healthy populations at recommended doses,” but it is important to consult with your doctor before beginning supplementation.

Caffeine

Caffeine is a stimulant that is often included in pre-workout supplements, as it has been shown to benefit athletic performance for short-term high intensity exercise and endurance-based activities. [7] It is important to understand that these studies have been conducted with Olympic and competition athletes, and thus the average individual who exercises recreationally should consult with a doctor before using caffeine as a supplement. For high performance athletes, the International Olympic Committee recommends 3–6mg caffeine/kg of body weight consumed an hour before exercise. [7-8] Evidence also suggests that lower caffeine doses (up to 3mg/kg body weight, ~200 mg) taken before and during prolonged exercise can increase athletic performance. [9-10] Mechanistically, caffeine increases endorphin release, improves neuromuscular function, vigilance, and alertness, and reduces perception of exertion during exercise. [10-11] Despite some benefits from smaller doses, larger doses of caffeine (>=9mg/kg of body weight) have not been shown to increase performance, and may induce nausea, anxiety, and insomnia. [11] The U.S. Food and Drug Administration (FDA) considers 400 milligrams of caffeine to be a safe amount for daily consumption, but some pre-workout supplements may exceed this amount in asingle serving or fail to disclose the amount of caffeine they contain, so it is important to always review the label of any supplement before consumption. Caffeine powder is also marketed as a stand-alone pre-workout supplement, but the FDA has advised against using this product, as very small amounts may cause accidental overdose. Powdered caffeine has been linked to numerous deaths—a single tablespoon (10 grams) is a lethal dose for an adult, but the product is often sold in 100-gram packages. [12]

Creatine

Creatine is a naturally occurring compound found in skeletal muscle that is synthesized in the body from amino acids and can be obtained from red meat and seafood. In the body, it helps produce adenosine triphosphate (ATP), which provides energy for muscles. Creatine is a popular workout supplement marketed to increase athletic performance, especially for weight training. Research suggests that creatine supplementation increases muscle vmix 22.0.0.54 registration key - Activators Patch of creatine, which in turn can enhance exercise capacity and training adaptations in adolescents, younger adults, and older adults. [13] Specifically, these adaptations allow for individuals to increase training volume (e.g., the ability to perform more repetitions with the same weight), which in turn can lead to greater increases in lean mass and muscular strength and power. [14-16] Although the exact mechanisms through which creatine improves performance have not been identified with certainty, various theories have been investigated, including the potential for creatine to stimulate muscle glycogen levels. [17,14] Creatine supplementation is primarily recommended for athletes who engage in power/strength exercises (e.g., weight lifting), or for athletes who engage in sports involving intermittent sprints and other brief repeated high-intensity exercises (e.g., soccer, basketball). [13] The International Society of Sports Nutrition recommends an initial dosage of 5g of creatine monohydrate (~0.3g/kg body weight) four times daily for 5–7 days to increase muscle creatine stores; once muscle creatine stores are fully saturated, stores can be maintained by ingesting 3–5 g/day. [13] Many powdered creatine supplements recommend this regimen in the directions on their packages. The Society also notes that an alternative supplementation protocol is to ingest 3g/day of creatine monohydrate for 28 days. [13] While the scientific literature has generally found supplementation to be safe at these levels, [18] creatine may not be appropriate for people with kidney disease or those with bipolar disorder. It is important to consult a doctor before taking this supplement. Of note, creatine supplementation has been shown to increase total body water, which causes weight gain that could be detrimental to performance in which body mass is a factor, such as running. [14] The International Society of Sports Nutrition, the American Dietetic Association, and the American College of Sports Medicine have all published statements supporting creatine supplementation as an effective way of increasing high-intensity exercise capacity and lean body mass during training for high-performance athletes. [19-21;3]


Post-Workout Supplements

A variety of post-workout supplements are marketed to consumers to increase muscle mass through enhanced muscle repair, recovery, and growth. Below is a review of some of the most common ingredients in post-workout supplements.

Carbohydrates

Replenishing glycogen stores after a workout with sufficient carbohydrate intake is important for muscle recovery, and beginning the next workout with sufficient muscle glycogen stores has been shown to improve exercise performance. [22-24] However, normal dietary intake is typically sufficient to restore muscle glycogen stores after low-intensity exercises, such as walking, yoga, or tai chi (3–5 g carbohydrate/kg body weight per day), and even for moderate-intensity exercise, such as one hour or more of walking, jogging, swimming, or bicycling at modest effort (5–7 g carbohydrate/kg body weight per day). [24] Post-workout supplementation with carbohydrates and protein within 24–36 hours is only recommended following strenuous physical activity, which includes one hour or more of hard exercise such as interval training, running, swimming, bicycling, soccer, or basketball at a moderate to intense effort (where one can only carry on brief conversations or cannot speak); in this case, 6–12 g carbohydrates/kg body weight per day is recommended to be consumed after exercise to fully restore muscle glycogen stores. [24]

Protein

Recommended levels of daily protein intake for the general population (0.8 grams of protein for every kilogram of body weight, or about 7 grams of protein every day for every 20 pounds of body weight) are estimated to be sufficient to meet the needs of nearly all healthy adults. [25] Recommendations for protein supplementation during exercise vary based on the type of exercise being conducted: endurance training (e.g., long-distance bicycling) or resistance training (e.g., weight lifting). Very few studies have investigated the effects of prolonged protein supplementation on endurance exercise performance. A review conducted by the International Society of Sports Nutrition found that protein supplementation in the presence of adequate carbohydrate intake does not appear to improve endurance performance, but may reduce markers of muscle damage and feelings of soreness. [26] On the other hand, individuals who engage in high-intensity resistance training may benefit from increased protein consumption to optimize muscle protein synthesis required for muscle recovery and growth, but research is inconclusive, with the majority of studies investigating the effects of protein supplementation on maximal strength enhancement finding no benefit. [26] The extent to which protein supplementation may aid resistance athletes is highly contingent on a variety of factors, including intensity and duration of training, individual age, dietary energy intake, and quality of protein intake. For individuals engaging in strenuous exercise to build and maintain muscle mass, the International Society of Sports Nutrition recommends an overall daily protein intake of 1.4–2.0 g/kg of body weight/day. [26] This can be ingested in the form of protein foods or protein powder.

Powdered protein can come from a variety of sources, including eggs, milk(e.g., casein, whey), and plants (e.g., soybeans, peas, hemp). Some protein powders contain protein from multiple sources; for instance, a vegan option might include protein derived from peas, pumpkin seeds, sunflower seeds, and alfalfa. Protein powders are dietary supplements and are not regulated by the FDA for safety. They can often contain non-protein ingredients, including vitamins and minerals, thickeners, added sugars, non-caloric sweeteners, and artificial flavoring. If you choose to consume protein powder, it is important to read the nutrition and ingredient labels beforehand, as products may contain unexpected ingredients and large amounts of added sugars and calories.
 
Powders, concentrates, isolates, and hydrolysates: What’s the difference?
Powdered protein supplements come in multiple forms, the most common of which are protein powders, protein concentrates, protein isolates, and protein hydrolysates. Proportionally, protein powders contain the least amount of protein, and often have higher concentrations of carbohydrates and fats from the original protein source. For example, whey protein powder, which comes from cow’s milk, is comprised of 11-14.5% protein, 63-75% lactose (milk sugar), and 1-1.5% milk fat. [27] Protein concentrates contain proportionally more protein (60-80%), with the remaining 20-40% composed of carbohydrates and fat. Additional filtering of protein concentrates creates protein isolates, which contain 90-95% protein. Protein hydrolysates are created by combining heat, enzymes, and/or acid with protein isolates to break the bonds between amino acids, allowing the compound to be absorbed more quickly by the body.
 
Some sources of protein supplements:
  • Casein and whey are proteins found in cow’s milk; roughly 80% of milk proteins are casein, while the other ~20% are whey. [28] Both proteins should be avoided by people who have trouble digesting dairy. Casein and whey contain all essential amino acids and are easily absorbed by the body, but their speed of absorption differs. [29-30] Whey protein is water soluble and rapidly metabolized into amino acids. Casein, on the other hand, is not soluble in water and is digested more slowly than whey—when ingested, it forms a clotted gel in the stomach that provides a sustained slow release of amino acids into the bloodstream over several hours. [31] Studies examining protein supplementation for resistance training suggest that whey’s faster digestion could be beneficial for gains in skeletal muscle mass compared to casein in both young men and in trained bodybuilders. [32-33] Another study, however, found that both proteins resulted in increased amino acid concentrations in the body compared to a placebo, with no significant differences between casein and whey for amino acid uptake or muscle protein balance. [34] Due to casein’s slower rate of absorption, it is often touted on health blogs as being useful for weight loss because it could hypothetically promote fullness, especially if ingested before periods of fasting, such as before bed. However, multiple studies have found no clear evidence that casein is more effective than any other protein source for satiety or weight loss. [35-36]
  • Soy protein powder is derived from soybeans, and unlike many plant-based proteins, it contains adequate levels of all essential amino acids. It is a common alternative manycam crack 2018 - Free Activators milk protein for vegans or people with dairy sensitivities or allergies. Soy protein is absorbed fairly rapidly by the body, although it is not as bioavailable as animal-based proteins. One study found that soy protein promoted muscle protein synthesis significantly more than casein protein when consumed by healthy young men at rest and after leg resistance exercise, but that soy protein was inferior to whey protein in increasing muscle protein synthesis. [32] A review of studies on milk- and soy-based protein supplementation also found that whey protein was better able to support muscle protein synthesis compared to soy protein in younger and older adults. [37]
  • Pea protein powder is made from yellow split peas, and can be an option for vegans or people with allergies or sensitivities to soy or dairy. Pea protein is rich in eight of the nine essential amino acids; it is low in methionine, which can be obtained from other sources including rice and animal proteins. There is limited research on the effects of pea protein. One double-blind, randomized, placebo-controlled study found that men aged 18 to 35 years who ingested 50 grams of pea protein daily in combination with a resistance training program over 12 weeks experienced similar increases in muscle thickness compared to those who ingested the same amount of whey protein daily. [38] However, all participants experienced similar increases in muscle strength, with no significant difference between those who supplemented with pea protein, whey protein, or a placebo.
  • Hemp protein powder is derived from the seeds of the hemp plant. Although there is little research on the use of hemp protein powder as a workout supplement, it contains omega-3 fatty acids and a number of essential amino acids. However, it is not a complete protein, as it has relatively low levels of lysine and leucine. [39-40]

Branched-Chain Amino Acids (BCAAs)

Three out of the nine essential amino acids have a chemical structure involving a side-chain with a “branch”, or a central carbon atom bound to three or more carbon atoms. These three amino acids, leucine, isoleucine, and valine, are called branched-chain amino acids (BCAAs). They can be obtained from protein-rich foods such as chicken, red meat, fish, and eggs, and are also sold as dietary supplements in powdered form. BCAAs are key components of muscle protein synthesis, [41] and research has shown that leucine in particular drives protein synthesis and suppresses protein breakdown. [42-43] Although short-term mechanistic data suggests that leucine plays an important role in muscle protein synthesis, [44] longer-term real-world trials do not support BCAAs as useful workout supplements. For example, a trial of leucine supplementation during an 8-week resistance training program did not result in increased muscle mass or strength among participants. [45] Studies have generally failed to find performance-enhancing effects of BCAAs such as accelerated repair of muscle damage after exercise; although ingestion of BCAAs is unlikely to be harmful, research does not support the health effects touted by supplement companies. [46]

Chocolate Milk

Although you may not think of it as a “supplement,” a number of pro athletes have begun to promote chocolate milk as an ideal post-workout beverage due to its combination of protein, carbohydrates, water, and electrolytes (in the form of sodium and calcium). A review of the effects of chocolate milk on post-exercise recovery found that chocolate milk provided similar or superior results compared to water or other sports drinks, [47] while another review found that cleanmymac x activation number keygen - Crack Key For U chocolate milk was an effective supplement to spur protein synthesis and glycogen regeneration. [48] However, the authors noted that evidence is limited and high-quality clinical trials with larger sample sizes are warranted. Of note, many studies of chocolate milk as a post-workout supplement are sponsored by the dairy industry, which may introduce bias. Chocolate milk generally contains high amounts of added sugars and saturated fat, and is likely most useful for athletes conducting high-intensity exercise for multiple hours a day, such as professional swimmers competing in the Olympics. However, for most individuals conducting moderate-intensity physical activity, such as an hour of jogging or bicycling, water is a healthier alternative as a post-workout beverage.

Electrolytes

Many supplements include electrolytes, which are chemicals that conduct electricity when mixed with water, and include sodium, potassium, and calcium. Electrolytes are important for hydration and the regulation of nerve and muscle function; for example, calcium, sodium, and potassium all work together to help muscles contract properly. The body loses electrolytes through sweating, so sports drinks (which typically contain carbohydrates/sugar and electrolytes) and other electrolyte supplements are often marketed as being necessary after a workout. However, the American College of Sports Medicine has asserted that there is little evidence of any difference in performance between those who drink beverages containing carbohydrates and electrolytes compared to those who drink plain water after exercising for less than one hour. [49] Sports drinks and other electrolyte supplements are generally only appropriate for people exercising vigorously for more than an hour, especially if causes them to sweat heavily. Learn more about sports drinks here.

Bottom Line

Workout supplements such as caffeine and creatine may be used to enhance exercise performance for regular high-intensity, strenuous physical activity, such as training to run a marathon or power lifting. However, a healthy diet with adequate amounts of healthy carbohydrates, protein, and water is sufficient to fuel the body for low to moderate amounts of physical activity, such as an hour of jogging or bicycling. As workout supplements are not regulated for safety, you should consult with a doctor before incorporating them into your exercise routine and discuss if there are any potential contraindications if you have existing medical conditions.

Related

Terms of Use

The contents of this website are for educational purposes and are not intended to offer personal medical advice. You should seek the advice of your physician or other qualified health provider with any questions you may have regarding a medical condition. Never disregard professional medical advice or delay in seeking it because of something you have read on this website. The Nutrition Source does not recommend or endorse any products.

Источник: https://www.hsph.harvard.edu/nutritionsource/workout-supplements/

Open Access

Peer-reviewed

  • Zhe Zhao,
  • Riku Fagerlund,
  • Helena Tossavainen,
  • Kristina Hopfensperger,
  • Rishikesh Lotke,
  • Smitha Srinivasachar Badarinarayan,
  • Frank Kirchhoff,
  • Perttu Permi,
  • Acid properties - Crack Key For U Sato,
  • Daniel Sauter,
  • Kalle Saksela
PLOS

x

?

This is an uncorrected proof.

Abstract

The accessory protein Nef of human and simian immunodeficiency viruses (HIV and SIV) is an important pathogenicity factor known to interact with cellular protein kinases and other signaling proteins. A canonical SH3 domain binding motif in Nef is required for most of these interactions. For example, HIV-1 Nef activates the tyrosine kinase Hck by tightly binding to its SH3 domain. An archetypal contact between a negatively charged SH3 residue and a highly conserved arginine in Nef (Arg77) plays a key role here. Combining structural analyses with functional assays, we here show that Nef proteins have also developed a distinct structural strategy—termed the "R-clamp”—that favors the formation of this salt bridge via buttressing Arg77. Comparison of evolutionarily diverse Nef proteins revealed that several distinct R-clamps have evolved that are functionally equivalent but differ in the side chain compositions of Nef residues 83 and 120. Whereas a similar R-clamp design is shared by Nef proteins of HIV-1 groups M, O, and P, as well as SIVgor, the Nef proteins of SIV from the Eastern chimpanzee subspecies (SIVcpzP.t.s.) exclusively utilize another type of R-clamp. By contrast, SIV of Central chimpanzees (SIVcpzP.t.t.) and HIV-1 group N strains show more heterogenous R-clamp design principles, including a non-functional evolutionary intermediate of the aforementioned two classes. These data add to our understanding of the structural basis of SH3 binding and kinase deregulation by Nef, and provide an interesting example of primate lentiviral protein evolution.

Author summary

Viral replication depends on interactions with a plethora of host cell proteins. Cellular protein interactions are typically mediated by specialized binding modules, such as the SH3 domain. To gain access to host cell regulation viruses have evolved to contain SH3 domain binding sites in their proteins, a notable example of which is the HIV-1 Nef protein. Here we show that during the primate lentivirus evolution the structural strategy that underlies the avid binding of Nef to cellular SH3 domains, which we have dubbed the R-clamp, has been generated via alternative but functionally interchangeable molecular designs. These patterns of SH3 recognition depend on the amino acid combinations at the positions corresponding to residues 83 and 120 in the consensus HIV-1 Nef sequence, and are distinctly different in Nef proteins from SIVs of Eastern and Central chimpanzees, gorillas, and the four groups of HIV-1 that have independently originated from the latter two. These results highlight the evolutionary plasticity of viral proteins, adobe photoshop 2020 crack reddit - Free Activators have implications on therapeutic development aiming to interfere with SH3 binding of Nef.

Citation: Zhao Z, Fagerlund R, Tossavainen H, Hopfensperger K, Lotke R, Srinivasachar Badarinarayan S, et al. (2021) Evolutionary plasticity of SH3 domain binding by Nef proteins of the HIV-1/SIVcpz lentiviral lineage. PLoS Pathog 17(11): e1009728. https://doi.org/10.1371/journal.ppat.1009728

Editor: Raul Andino, University of California San Francisco, UNITED STATES

Received: June 17, 2021; Accepted: October 28, 2021; Published: November 15, 2021

Copyright: © 2021 Zhao et al. This is an open access article distributed under the terms of Wondershare Recoverit 9.7.2.12 Crack Plus Registration Key Free2021` Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: KS was funded by the Helsinki University Central Hospital Research Council grant TYH2017248 and by the Jane and Aatos Erkko Foundation (JAES2016). PP was supported by grants from the Academy of Finland (323435) and Jane and Aatos Erkko Foundation (JAES2019). DS was supported by the Heisenberg Program (SA 2676/3-1) and the Priority Program SPP1923 (SA 2676/1-2) of the German Research Foundation (DFG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Primate lentiviruses comprise human immunodeficiency viruses (HIV-1 and HIV-2), as well as more than 40 simian immunodeficiency viruses (SIVs) infecting non-human primate species from sub-Saharan Africa. HIV-1 groups M, N, O, and P originate from four independent transmissions of SIV from Central chimpanzees (SIVcpzP.t.t.) and gorillas (SIVgor) to humans, whereas HIV-2 groups A to I are derived from nine zoonotic transmissions of SIV from sooty mangabeys (SIVsmm) (reviewed in [1]). SIVsmm was also transmitted to macaques, giving rise to SIVmac. The global AIDS pandemic is largely caused by HIV-1 group M strains, which are further divided into 10 subtypes (A, B, C, D, F, G, H, J K, and L), as well as several sub-subtypes and circulating recombinant forms [2–4].

The viral protein Nef is a multifunctional accessory factor encoded by all primate lentiviruses. While nef-defective HIV and SIV are replication-competent, infections with such viruses are associated with low viral loads and no or delayed pathogenesis in humans or experimentally infected macaques [5]. Nef itself exhibits no enzymatic activity but instead modulates host cell function by interacting with a plethora of other proteins to hijack cellular signaling and membrane trafficking pathways [6–8].

A function of HIV-1 Nef that is well understood at the molecular and mechanistic level is its interaction with Hck, a member of the Src-family tyrosine kinases. Nef tightly binds to the SH3 domain of Hck, thereby shifting it from an intramolecular autoinhibitory state into a catalytically active conformation [9,10]. Hck plays an important role in regulating reason 10.4 crack - Activators Patch and effector functions of macrophages, the second main target cell population of HIV-1 besides CD4+ T cells [11,12]. Many Nef functions in other cell types including T lymphocytes, (e.g., MHC-I downregulation [13], interfering with Lck localization and immunological synapse formation [13], and reorganization of the actin cytoskeleton [14,15]) are also strictly dependent on its SH3 binding capacity. However, the relevant SH3 proteins in these cells do not bind to Nef as tightly as Hck, and have therefore remained less well characterized [7].

The discovery of the Nef-Hck interaction provided the first example of pathogen takeover of host cell signaling via SH3 domain ligand mimicry [16]. Src Homology 3 (SH3) domains are short (~60 aa) modular protein units specialized for mediating protein interactions via proline-rich core binding sites in the target proteins, and are ubiquitous (~300 in human) in eukaryotic proteins involved in regulation of cell behavior [17,18].

Studies on the Nef-SH3 complex also revealed a new paradigm for the structural basis of SH3 binding: It could be shown that docking of the proline-rich (PxxP) peptide of Nef by the SH3 core binding interface is assisted by further molecular contacts of the so-called SH3 RT-loop region to provide additional specificity and affinity to this interaction [9]. The amino acid residues that form a binding pocket for the RT-loop of the Hck-SH3 domain are highly conserved in HIV-1 and HIV-1-like Nef proteins, and provide them with a capacity for strong binding to Hck [9,19]. Nef proteins from the HIV-2/SIVsmm groups lack these residues and hence Hck binding capacity, but as evidenced by the SIVmac293-Y113W/E117T/E118Q triple mutant [20], can acquire this function upon introduction of just a few key residues from HIV-1 Nef.

We have recently shown that Hck activation by Nef leads to Raf/MAPK pathway activation and triggers the secretion of proinflammatory cytokines [19,21]. In agreement with their enhanced affinity for Hck [9,20], we found that only HIV-1/SIVcpz type Nef proteins but not HIV-2/SIVsmm type Nef proteins show this function. The current study was incited by our observation that an HIV-1 group N Nef (clone YBF30) was unable to bind and activate Hck and the Raf/MAPK pathway for reasons that could not be readily explained by its amino acid sequence. Our investigation into this issue led to the discovery of a structural arrangement that we have termed the arginine (R)-clamp and characterized here. We show that different but functionally equivalent R-clamps have emerged in the HIV-1/SIVcpz lineage to coordinate Hck SH3 domain binding by Nef. This finding further highlights the importance of Nef-mediated SH3 binding and the enormous plasticity of primate lentiviral accessory proteins.

Results

In our previous studies we examined the capacity of Nef to activate Hck by monitoring tyrosine phosphorylation of paxillin, a prominent substrate for Src family tyrosine kinases, and by measuring the induction of AP-1-regulated reporter gene expression following Hck-activated MAPK signaling [19,21]. When additional Nef proteins from HIV-1 groups N, O, and P were examined (Fig 1), we unexpectedly noticed that the Nef protein of HIV-1 N YBF30 (AJ006022) was unable to induce paxillin phosphorylation or AP-1 activity despite sharing the 72PxxPxR77 motif (numbering based on the HIV-1 Nef consensus) known to be important for Hck binding by HIV-1/SIVcpz type Nefs (Fig 2). Importantly, YBF30 Nef was clearly expressed in these cells (Fig 1A), and as also shown previously [22] was functional when tested for its capacity to downregulate cell surface expression of CD4 (Figs 1C and S1).

thumbnail
Download:

Fig 1. Hck-activating potential of selected HIV-1 Nefs.

(A) Hck-expressing HZ-1 cells were co-transfected with vectors for paxillin and Nef from the indicated HIV-1 group M, N, O or P clones, or mutants thereof. Lysates of the transfected cells were analyzed by Western blotting using antibodies against paxillin (PXN), phosphorylated paxillin (pPXN) and Nef. (B) HZ-1 cells were transfected with an AP-1 transcription factor-driven luciferase reporter alone (Control) or together with the indicated Nefs variants. Luciferase activity was measured in cells harvested 24 h post-transfection, and normalized to the corresponding control sample that was set to 1. The data shown are derived from three independent experiments, with SE indicated by error bars. The amino acid at position 120 of each Nef protein is indicated, aromatic residues are shown as a black 1-letter symbol in a white sphere, and isoleucine as a white letter in a black sphere. (C) Jurkat T cells stably expressing CD4 were infected with lentiviral vectors expressing GFP alone (No Nef) or together with plasmids expressing the indicated Nefs. CD4 down-regulation by Nef was measured using flow cytometry 48 h after lentiviral transduction, and histograms illustrating cell surface levels of CD4 among the GFP positive Jurkat cells are shown. Original dot plots are shown in S1 Fig.

https://doi.org/10.1371/journal.ppat.1009728.g001

thumbnail
Download:

Fig 2.

HIV-1 M consensus Nef amino acid sequence (A) and an alignment of the conserved central region (underlined in A) of selected Nef proteins from different primate lentiviruses (B). The key residues of the core SH3 docking site (PxxPxR motif), including R77, are highlighted in light blue. Residues involved in a binding pocket for the Hck SH3 domain RT-loop that are conserved in HIV-1 and HIV-1-like Nef proteins are highlighted in yellow. Residues at positions 83 and 120 forming the R-clamp coordinating the positioning of R77 are shown in bold and colored according to their side chain properties (small in green, hydrophilic in blue, aliphatic or methionine in red, aromatic in brown). The names of HIV/SIV strains from which Nef proteins were included in this study are shown on the left.

https://doi.org/10.1371/journal.ppat.1009728.g002

To examine if this is a characteristic of several HIV-1 group N viruses we tested three additional HIV-1 N Nef proteins from the strains 02CM-DJO0131 (AY532635), YBF106 (AJ271370), and S4858 (KY498771). However, we found that all of them were fully competent for Hck activation, and induced paxillin phosphorylation and AP-1 activity as efficiently as Nef from the widely PureVPN Offline Installer HIV-1 group M strain SF2 (Fig 1).

Since the core SH3 docking motif (72PxxPxR77) as well as the key RT-loop accommodating residues, including F90, W113, T117, and Q118, were conserved in YBF30 Nef (see Fig 2) we had a more detailed look at the original X-ray structure of the HIV-1 Nef-SH3 complex (1EFN; [9]), which directed our attention to Nef residue 120. This position is occupied by an aromatic residue (tyrosine or phenylalanine) in virtually all HIV-1 M Nef proteins, while an isoleucine residue is found in this position in YBF30 Nef (Fig 2). In the 1EFN structure, Y120 can be seen to coordinate the Nef-SH3 interaction by buttressing the side-chain of the R77 in the Nef 72PxxPxR77 motif together with the SH3 residue W37 (SH3 numbering according to [18]) in order to stabilize the close positioning of R77 with the acidic SH3 residue D17 to form a critical salt bridge with it (Fig 3A and 3B). We termed this steric guiding of the Nef residue R77 into close proximity of D17 in Hck-SH3 the “arginine (R)-clamp”, and hypothesized that the failure of YBF30 Nef to activate Hck is due to the failure of its Ile120 residue to from a functional R-clamp (Fig 3C).

thumbnail
Download:

Fig 3. The arginine clamp.

(A) Structure of the HIV-1 Nef (green)–SH3 (pink) complex (PDB ID 1EFN) highlighting critical residues in the interaction interface. (B) Close up view of the R clamp, stabilized by stacked Y-R-W side chains and a salt bridge between R77 (Nef) and D17 (SH3) side chains. (C) In the Y120I structure, one side of the R77 guanidinium plane is lacking non-bonded interactions. (D) In the A83M/Y120I, structure the long methionine side chain re-establishes non-bonded contacts for R77. The presented mutated complex structures were created by replacing Y120 or Y120 and A83 in UCSF Chimera [45] and minimizing the structures with AMBER [46].

https://doi.org/10.1371/journal.ppat.1009728.g003

In support of this hypothesis, we found that YBF30 Nef became fully competent for Hck activation when its I120 residue was replaced by a tyrosine (YBF30 I120Y) (Fig 1). We then further investigated this idea by changing the Y120 residue in the Nef protein from the HIV-1 M laboratory strain SF2 into an YBF30-like isoleucine (Nef mutant SF2 Y120I) or into a phenylalanine, the other commonly found residue at this position (Nef mutant SF2 Y120F). While the conservative Y120F amino acid change did not affect Hck activation, the Y120I mutation recapitulated the failure of YBF30 Nef to stimulate paxillin phosphorylation and AP-1 activity, without substantially compromising protein expression levels or CD4 downregulation (Fig 1). Thus, we conclude that the lack of a functional R-clamp due to the I120 residue likely explains the inability of YBF30 Nef to activate Hck.

Analysis of additional HIV-1 group N Nefs revealed that in 5 out of the total of 12 sequences that are available in databases the position 120 was occupied by Ile (Table 1). This is in stark contrast to the Nef proteins from the three other HIV-1 groups M, O, and P, in which residue 120 is invariably Y or F. We had access to another I120-containing HIV-1 N Nef namely 2693BA (GQ925928), and went on to examine its ability to activate Hck. Having already established a strict correlation between paxillin phosphorylation and AP-1 activity as markers of Hck activation (Fig 1 and [19]), we chose to focus on using AP-1-driven reporter gene expression as the read-out in our further studies. Unexpectedly, we found that despite carrying the I120 residue 2693BA Nef had an undiminished capacity to activate Hck (Fig 4). In order to understand this finding, we returned to the Nef-SH3 structure 1EFN, and noted the involvement of Nef residue 83 in contacting the critical R-clamp residue 120 (Fig 3). Residue 83 is an amino acid with a tiny (Ala or Gly) or a hydrophilic side-chain (Gln, Ser, Asp, or Glu) in virtually all Nef proteins that have an aromatic (Phe or Tyr) residue at position 120. However, in 2693BA Nef position 83 is occupied by methionine, a residue with a large hydrophobic/aliphatic side-chain. We therefore hypothesized that M83 might compensate for the presence of isoleucine instead of a planar aromatic residue at position 120 (Figs 3D and S4), and that the M83/I120 residue pair of 2693BA Nef might be functionally equivalent to the A83/F120 pair found in the Hck-activating HIV-1 N Nef proteins 02CM-DJO0131 and YBF106.

thumbnail
Download:

Fig 4. Role of residue 83/120 pairing in the HIV-1 N Nef R-clamp.

(A) HZ-1 cells were transfected with an AP-1-dependent luciferase reporter alone (Control) or together with wild type (WT) versions of YBF30 or 2693BA Nef, or mutants thereof carrying reciprocal amino acid changes of residue 83 (YBF30 Q83M and 2693BA M83Q). Luciferase activity was measured from cells 4 h post-transfection, and normalized to the corresponding control sample that was set to one. The data shown are derived from three independent experiments, with SE indicated by error bars. The amino acid combinations at positions 83 and 120 are indicated here and in the figures below as single-letter symbols and color-coded according to the residue classification shown in Table 1. Specifically, small (G or A) or hydrophilic residues (D, E, Q, or S) at position 83 are shown as a black 1-letter symbol in a white sphere (here Q83 in YBF30), whereas aliphatic or methionine residues at this position are shown as a white letter in a black sphere (here M83 in 2693BA). At position 120, aromatic residues (F or Y) are shown as a black letter in a white sphere (not found in YBF30 or 2693BA), whereas isoleucine is shown as a white letter in a black sphere (here I120 in YBF30 and 2693BA). (B) CD4 downregulation by Nef in stably transduced Jurkat T cells was examined as in Fig 1C. Original dot plots are shown in S2 Fig.

https://doi.org/10.1371/journal.ppat.1009728.g004

To test this idea, we swapped residues 83 between the I120-containing 2693BA and YBF30 Nef proteins. In line with our hypothesis, this reversed the differences in the Hck-activating capacity of these HIV-1 N Nefs, such that Nef-2693BA lost its ability to stimulate AP-1 activity, whereas YBF30 Nef became fully competent for this function (Fig 4). Thus, we conclude that Nef residues 83 and 120 appear to function together to form the R-clamp that coordinates Hck-SH3 binding by Nef, and that different combinations of residue pair 83/120 have evolved for building a functional R-clamp in HIV-1 group N Nefs.

Whereas among the Nef proteins of HIV-1, Ile can be found at position 120 only in group N viruses, we noted that several SIVcpz Nefs also contain Ile120 (Table 1). To test if our conclusions regarding the alternative R-clamp designs hold true also for SIVcpz Nef proteins, we examined five of these, three from SIV of the chimpanzee subspecies Pan troglodytes troglodytes (P.t.t.), namely MB897 (EF535994), CAM5 (AJ271369), and EK505 (DQ373065), and two from Pan troglodytes schweinfurthii (P.t.s.), namely Tan2 (EF394357) and Nok5 (AY536915).

We found that similar to the HIV-1 N YBF30 Nef, some of these SIVcpz Nefs (Cam5 and EK505) failed to activate Hck, whereas others (MB897, Nok5, and Tan2) were functional in this regard although all were able to downregulate CD4 (Fig 5). When the Hck activating capacity was compared with the residue 83/120 composition of these SIVcpz alleles, a perfect agreement was found with the structural R-clamp principles deduced from the results obtained with HIV-1 N Nefs. All SIVcpz Nef proteins that were competent for Hck activation contained a “permissible” [83] + [120] residue combination defined as [tiny/hydrophilic] + [Phe/Tyr] or [aliphatic] + [Ile], whereas both of the two non-activating SIVcpz Nef proteins contained a mixed pattern of amino acid classes at these positions (Figs 2 and 5).

thumbnail
Download:

Fig 5. Free folder lock software architecture of SIVcpz Nef proteins.

(A) HZ-1 cells were transfected with an AP-1-luciferase reporter alone (Control) or together with wild-type or the indicated mutants of Nef from SIV strains from Pan troglodytes troglodytes (MB897, Cam5, and EK505) or Pan troglodytes schweinfurthii (Nok5 and Tan2). Luciferase activity in the transfected lysates was analyzed as in Fig 1C. The data shown are derived from three independent experiments, with SE indicated by error bars. The amino acid combinations at positions 83 and 120 are indicated as single-letter symbols and color-coded as explained in Fig 4. (B) CD4 downregulation by Nef in stably transduced Jurkat T cells was examined as in Fig 1C. Original dot plots are shown in S3 Fig.

https://doi.org/10.1371/journal.ppat.1009728.g005

To further prove this concept, we showed that the failure of Cam5 and EK505 Nef to activate Hck could indeed be corrected by introducing a tyrosine residue at position 120 in these proteins, thereby providing them with an R-clamp residue combination 83 [hydrophilic] + 120 [Phe/Tyr]. Moreover, an E83M mutation (resulting in an 83 [aliphatic] + 120 [Ile] residue combination) conferred this activity to EK505 Nef. On the other hand, introducing a hydrophilic residue at position 83 of Nok5 Nef while maintaining its I120 residue (Nok5 I83Q) led to a complete loss of its Hck-activating potential, as predicted by our model on the compatible and non-compatible R-clamp residue combinations.

When the my mac cleaner 3 83/120 composition was analyzed in a comprehensive survey of HIV-1/SIVcpz/SIVgor Nef sequences, it was interesting to observe that the different evolutionary lineages of primate lentivirus are associated with distinct R-clamp design strategies. In Table 1, these strategies have been grouped into four categories (I–IV) based on the amino acid side chain properties explained above, and their occurrence has been calculated as a percentage of all Nef sequences found for each lentivirus lineage. Whereas HIV-1 M, HIV-1 O, HIV-1 P, and SIVgor viruses fall almost exclusively into category I, HIV-1 N and SIVcpz viruses show more heterogeneity. Interestingly, however, all SIVcpz Nef acid properties - Crack Key For U from the chimpanzee subspecies P.t.s. fall into category IV, while all SIVcpz(P.t.t.) Nef proteins are found in categories I (67%) or III (33%). HIV-1 N Nef proteins share the R-clamp design patterns with SIVcpz(P.t.t.) dxtory portable - Free Activators SIVcpz(P.t.s.), and can be found spread between categories I, III, as well as IV. Finally, exclusively HIV-1 M Nef proteins can be found in category II, which includes a small but significant proportion (2.6%) of all the 5221 HIV-1 M Nef protein sequences in our survey.

Of note, the relative distribution of HIV-1 M Nef sequences into categories I to IV is not even between different viral subtypes. Whereas subtype B that constitutes more than half of all available HIV-1 M Nef sequences rarely falls in I120-containing categories III (0.31%) or IV (0.03%), this is much more common among subtype D and G Nef proteins, of which 2.91% and 2.59%, respectively belong to categories III or IV. And although only four of out the total 5221 HIV-1 M Nef sequences belong to category IV, two of these can be found among the 206 subtype D Nef sequences. On the other hand, while category II covers 2.6% of total HIV-1 M Nef sequences, this is the case for as many as 15.6% of subtype G and 7.1% of subtype H, but only 0.5% of subtype A strains.

Based on the experiments presented above it was inferred that Nef proteins in categories I, II, and IV can bind Hck to stimulate its tyrosine kinase activity, whereas category III Nef proteins with a “mixed” R-clamp residues pattern fail to do so. To further substantiate the generality of this conclusion, we introduced several additional individual and combinatorial 83/120 residue changes into the model Nef protein of HIV-1 M SF2 according to the classification presented in Table 1. We found that a Gln, Glu, or Ser residue could indeed all be introduced at position 83 of SF2-Nef without losing its Hck-activating function, as long as an aromatic residue was maintained at position 120 (Fig 6; SF2 A83Q, A83E, and A83S). Thus, all these different category II configurations could support a functional R-clamp when artificially introduced into SF2-Nef.

thumbnail
Download:

Fig 6. Comprehensive testing of R-clamp residue pairing rules by mutagenesis of SF2-Nef.

Nef-induced AP-1 activity in cells transfected without Nef (Control) or with the indicated wild-type or mutant versions of SF2 Nef was analyzed as in Fig 1B. The amino acid combinations at positions 83 and 120 of these SF2 Nef variants are indicated as single-letter symbols that are color-coded as explained in Fig 4.

https://doi.org/10.1371/journal.ppat.1009728.g006

By contrast, and again in full agreement with our R-clamp model, combination of these mutations with an Y120I change to introduce a category III “mixed” residue 83/120 pattern into SF2 (Q83S/Y120I, A83S/Y120I, and A83E/Y120I) resulted in all three cases in the loss of Hck activation. Finally, the disrupted Hck-activating function of the single residue 120 mutant SF2 Y120I could be fully rescued via an additional introduction of Met, Ile, or Leu at position 83 to generate artificial category IV-like SF2 Nef double mutants A83M/Y120I (“2693BA-like”), A83I/Y120I (“Nok5-like”), and A83L/Y120I (“Tan2-like”).

To confirm that the effects of the Nef R-clamp mutations on the observed cellular changes downstream of Hck activation were indeed due to altered formation of physical Nef-Hck complexes, we examined how wild-type and R-clamp mutated Nef proteins coprecipitated with Hck-p59 from transfected cells. As shown in Fig 7A, association with Hck was observed only for Nef proteins having a functional R-clamp, whereas category III R-clamp Nef proteins were as defective in this regard as the Nef-AxxA mutant carrying a fully disrupted SH3-binding motif.

thumbnail
Download:

Fig 7. Role of R-clamp residues 83/120 in intracellular Hck-Nef complex formation.

HEK293 cells were transfected with Myc-tagged wild-type HIV-1 M SF2 Nef (WT) or its mutants including Y120I, A83L-Y120I, P76A-P78A (AxxA), A83Q, and A83Q-Y120I) together with biotin acceptor domain-tagged wild-type Hck-p59 (WT) (A.) or its SH3 mutant Hck-C3 (B.). The Nef-contacting RT-loop residues centered around the critical SH3 amino acid 13 (Ile in WT and Asp in C3) are shown on top of these figures. Best image converter - Free Activators of transfected cells were subjected to anti-Myc immunoprecipitation followed by Western blotting analysis of the immune complexes using labeled streptavidin (top panels). Equal Hck and Nef expression in the total lysates was confirmed by labeled streptavidin (Hck) (middle panels) or an anti-Myc (Nef) antibody (bottom panels).

https://doi.org/10.1371/journal.ppat.1009728.g007

In addition to its Class II binding motif (PxxPxR) a hydrophobic pocket in the core domain Nef is important for the Hck interaction by making contacts with the RT-loop region of Hck SH3 [9], in particular the isoleucine residue 13 (universal SH3 numbering according to [18]). Because the R-clamp residues 83 and 120 are part of the compact core of Nef, we wanted to exclude the possibility that mutations of these residues might influence Hck binding by disrupting the stabilizing contacts between Nef and I13 of Hck SH3. To this end, we constructed modified versions of Hck-p59 having artificial SH3 domains containing RT-loops that lack I13 and interact with Nef using diverse non-Hck-like molecular strategies [23,24]. Despite their dissimilar contacts with the hydrophobic pocket of Nef, all of these SH3-modified Hck proteins shared the wild-type Hck-like dependence on a functional R-clamp for interacting with Nef. These data are shown in Fig 7B for the Hck mutant C3 where the native RT-loop residues EAIHHE have been replaced with YSDFPW (notably containing aspartate, a charged hydrophilic residue at the SH3 position 13 instead of an isoleucine), and similar data for additional two SH3-modified Hck proteins are provided in S5 Fig

To examine the role of the Nef R-clamp in cellular system that is more relevant for HIV biology than HEK293 cells, we tested the capacity of different lentivirally to induce cellular activation in vitro differentiated THP-1-derived human macrophagic cells (Fig 8). This model for macrophage activation depends on SH3 binding by Nef, and is based on monitoring of the activation of the MAPK signaling cascade using phosphorylation of Erk1/2 as the read-out [19]. As evident from two independent experiments involving a set of YBF30 (HIV-1 N) and Nok5 (SIVcpz(P.t.s)) Nef proteins (Fig 8A) or a panel of SF2 Nef variants (Fig 8B), the capacity to activate macrophages and induce their Erk phosphorylation over the steady-state background levels required a functional R-clamp configuration.

thumbnail
Download:

Fig 8. Role of the R-clamp in activation of in vitro differentiated macrophages by Nef.

The indicated wild-type or mutant Nef proteins from HIV-1 M (panels A. and B.), HIV 1 N (panel A.), or SIVcpz(P.t.s) (panel A.) were expressed via lentiviral transduction in M1-like macrophages differentiated from THP-1 cells, followed by Western blotting analysis to compare the expression of Nef, total ERK1/2, and phosphorylated ERK1/2 in these cells or control macrophages transduced with an empty lentivector (-).

https://doi.org/10.1371/journal.ppat.1009728.g008

Finally, we also studied the role of the R-clamp in another well-established cellular function of Nef, namely enhancement of HIV-1 particle infectivity [25,26]. The underlying mechanism involving counteraction of the SERINC5 restriction factor is not SH3 dependent [27]. However, disruption of the PxxP motif has been associated with a modestly lowered capacity of Nef to increase virion infectivity [28]. When a representative panel of HIV-1 M, HIV-1 N, and SIVcpz(P.t.s) Nef alleles with wild-type or altered residues 83/120 were tested for their ability to increase the infectivity of HIV-1 particles (S6 Fig) no apparent differences were observed. This supports the idea that enhancement of HIV-1 infectivity does not depend on SH3 binding by Nef, and confirms our conclusion based on CD4 downregulation that R-clamp inactivating mutations do not universally affect Nef functionality.

Together, our results provide strong further proof to our conclusions on the 83/120 residue combinations that are required for Hck binding and activation, and demonstrate interesting structural and evolutionary plasticity in organizing a set of molecular interactions via an R-clamp principle to enable Nef to tightly bind the SH3 domain of Hck.

Discussion

Apart from the regulation of trafficking of CD4 and other membrane proteins, the majority of all cellular functions described for Nef depend on its SH3 domain binding capacity, and are lost if the highly conserved consensus SH3 ligand motif PxxPxR of Nef is mutated [7]. Here we show that Nef proteins of HIV-1 and closely related SIVs have evolved a sophisticated molecular mechanism that we have termed the R-clamp in order to coordinate their SH3 binding. This term was coined due to buttressing of the arginine residue of the Nef PxxPxR motif (R77) by the coordinated action of the Nef residues 83 and 120 together with a tryptophan residue (W37) that is conserved in almost all SH3 domains. This places R77 of Nef into close proximity with another highly conserved SH3 residue (D17) to form a salt bridge that stabilizes the Nef/SH3 complex. Thus, docking of proline-rich ligand peptides presented by native proteins, such as Nef, can involve an additional level of structural complexity for tuning of SH3 binding affinity and specificity that is not evident from studies with isolated SH3 binding peptides.

Our studies have focused on the SH3-mediated interaction between Nef and the tyrosine kinase Hck leading to enzymatic activation of Hck, which we have monitored based on induction of downstream signaling events, including paxillin phosphorylation as well as triggering of the Raf/MAPK cascade and subsequent AP-1-regulated gene expression. Nef/Hck interaction is thought to play an important role in HIV infection of cells of the myeloid lineages, such as macrophages, and is an especially tractable study system because of the high affinity of Hck SH3 domain for Nef [19]. This is due to the additional affinity brough into this interaction by tertiary contacts outside of the canonical SH3 ligand binding surface and involving the tip of the RT-loop in Hck SH3 [9,23]. Because of the lack of such RT-loop contacts, SH3 domains of other host cell proteins bind to Nef with a lower affinity resulting in more transient interactions that have remained less well characterized, but appear to include at least Lck [29], Vav [30], PACS-1 [31], Btk, and Itk [32]. Nevertheless, similar to Hck-binding, most of the other SH3 interactions by Nef are also expected to depend on formation of a salt bridge between R77 of Nef and acidic (D or E) SH3 residue 17. Thus, all such interactions, including the SH3-mediated complexes that are relevant for Nef in T lymphocytes depend on a functional R-clamp. However, it cannot be ruled out that despite containing a canonical Class II SH3 binding motif (PxxPxR) Nef (including category III) might also engage in unorthodox interactions with (an) atypical SH3 domain(s) that would not involve R-clamp coordination of R77.

Interestingly, we found that in different lentiviral Nef proteins the R-clamp has been assembled based on alternative designs, which depend on the combination of amino acids at positions 83 and 120. Moreover, the preferred 83/120 residue combinations are dissimilar in different lineages of HIV-1, SIVcpz, and SIVgor (Table 1). While the vast majority of HIV-1 group M, O and P Nefs belongs to category I, HIV-1 N Nefs are more heterogenous and fall into categories I or IV (harboring an active R-clamp) or inactive category III (Fig 9). Why would about 25% of all HIV-1 N Nefs harbor an inactive R-clamp, whereas their counterparts from HIV-1 groups M, O, and P do not? Of note, SIVcpz(P.t.t.), the simian precursor of HIV-1, comprises category I and III Nefs. Based on the phylogenic analysis of primate lentiviral Nef sequences shown in Fig 9B, we propose that the zoonotic transmission that gave rise to HIV-1 group N involved a virus carrying a Nef with a category III R-clamp.

thumbnail
Download:

Fig 9. Putative cross-species transmission and evolution of R-clamp categories.

The pie charts on the left illustrate the relative fraction of each R-clamp category in SIVcpz, SIVgor and HIV-1 groups M, N, O and P. Putative cross-species transmission events are indicated by colored arrows. The lower part of the figure shows a phylogenetic analysis of selected primate lentiviral Nefs is shown. Inferred most recent common ancestors are indicated by stars. In both images, R-clamp categories I, II, III, and IV are shown in blue, yellow, grey and orange, respectively.

https://doi.org/10.1371/journal.ppat.1009728.g009

In line with this hypothesis, as indicated in Fig 9B the inferred common ancestor of HIV-1 group N viruses belongs to category III as its Nef protein harbors a combination of Q83 and I120 [33](GenBank accession: KP059120.1). Furthermore, the SIVcpz(P.t.t.) isolate EK505 (DQ373065), a close relative of HIV-1 group N viruses [34] also expresses a category III Nef. Thus, HIV-1 group N viruses may all be the result of a category III virus, and still be adapting and evolving into categories I and IV with functional R-clamps. In this regard it should be noted that group N most likely represents the evolutionarily youngest of all HIV-1 groups [35,36], and be appreciated that the three most recently isolated group N Nefs (JN572926, MF767262, KY498771), including that of N1_FR_2011, the only group N virus isolated outside Cameroon [37], belong to categories I or IV. A single amino acid change of residue 83 (like the EK505-E83M mutant created in this study; see Fig 5) would have been sufficient to move from category III to IV, whereas the path from category III to I would require changes in both residues 83 and 120, and thus follow an evolutionary trajectory via category II. None of the currently available HIV-1 N Nef sequences fall into the R-clamp category II, but given the scarcity (n = 12) of these sequences this does not mean that such HIV-1 N Nefs could still not exist.

In contrast to HIV-1 N, group M viruses almost exclusively fall into category I, suggesting that the zoonotic jump from SIVcpz(P.t.t) to HIV-1 M involved a virus with a category I Nef R-clamp. Indeed, the inferred most recent common ancestor of HIV-1 M Nefs (KP059118.1; KP059119.1; [33] belongs to category I. This is also true for the oldest known HIV-1 M Nef (M15896) isolated in 1976 [38]. Finally, the exclusive use of category I R-clamp by Nef proteins of SIVgor, HIV-1 O, and HIV-1 P would suggest that an R-clamp category I virus might have originally been transmitted from chimpanzee to gorilla, and subsequently in two

It is of interest to note that a subtype D virus of the HIV-1 M group has been previously shown to encode a Nef protein unable to activate Hck, and that this deficiency was mapped to the unusual presence of an isoleucine residue at the Nef position 120 [39]. This study suggested that Y120 would contribute to the hydrophobic pocket of Nef that accommodates the Hck SH3 domain RT-loop, whereas I120 could asc timetables 2020 crack keygen - Free Activators serve this function. However, these molecular contacts inferred based on our (NL4-3) Nef/SH3 X-ray structure (1EFN; [9]}) would be expected to only minor rather than critical for the Nef-Hck interaction, and a recent SH3 complex structure involving HIV-1 SF2 Nef [40] does not support such a role for Y120 at all. Moreover, our current data clearly show that category IV Nef proteins containing I120 do bind and activate Hck when paired with an appropriately matched R-clamp residue at the position 83. Thus, while the findings of Choi and Smithgall on Nef from HIV-1 ELI [39] agree with our data, they need to be interpreted in light of the R-clamp concept described here.

While the evolutionary scenarios discussed above may help to explain the occurrence of inactive category III Nefs in HIV-1 group N, it still remains unclear why about one third of all SIVcpz(Pt.t.) Nefs harbor a category III R-clamp. Furthermore, the reasons for the unique predominance of category IV Nefs in SIVcpz(P.t.s.) remain to be determined. Apart from the initial founder viruses that gave rise to the respective lentiviral lineages and species, the optimal R-clamp composition and the rate of R-clamp sequence evolution are probably determined by a complex balance between host-specific factors and sequence variation elsewhere in these viruses. To gain further insights, it would be interesting to examine a larger number of HIV-1 N Nef sequences over time and from different tissues from acid properties - Crack Key For U same individual to understand whether the R-clamp pattern is relatively fixed or subject to rapid evolution and quasispecies variation. In the latter case, because of the highly multifunctional nature of the Nef protein, a non-functional R-clamp might provide a selective advantage in certain anatomic locations or special circumstances that might occur during or immediately after the zoonotic jump from chimpanzee to man. On the other hand, as already noted, despite the virtually universal conservation of an intact Class II SH3 binding motif PxØPxR (where Ø is a hydrophobic residue) in all Nef proteins (including R-clamp category III), it could be speculated that while unable to interact with canonical SH3 domains, R-clamp category III Nef proteins might instead show unusual specificity for some atypical SH3 proteins, which could provide them with alternative cellular functions.

In any case, R-clamp sequence variation provides a fascinating example of evolutionary plasticity of a protein interaction interface and our findings suggest that the selection pressures that have shaped Nef during primate lentiviral evolution are different depending on the HIV/SIV lineage and their host species. The dynamic capacity of Nef for altering the molecular strategy of recognizing its key host cell interaction partners, such as SH3 domains, is also important to keep in mind in attempts to develop novel HIV eradication therapies that might target the immune evasion function of Nef.

Materials and methods

Reagents and cell lines

Mouse anti-Myc (sc-40) and rabbit anti-paxillin (sc-5574) were from Santa Cruz Biotechnology. Mouse anti-GAPDH (607902) was from Biolegend). Mouse anti-pY31 paxillin was from BD Biosciences. Rabbit anti phospho-p44/42 MAPK (ERK1/2) (9101) and rabbit anti- p44/42 MAPK (ERK1/2) (9102) antibodies were from Cell Signaling Technology. Alexa Fluor 647 conjugated anti-human CD4 antibody was from SouthernBiotech. IRDye680CW goat anti-mouse IgG, IRDye800CW Streptavidin, and IRDye800CW goat anti-rabbit IgG were from LI-COR Biotechnology. HEK293, HEK293T, THP-1, and Jurkat cells were obtained from ATCC. TZM-bl cells are a HeLa-derived reporter cell line and were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH, from John C. Kappes, Xiaoyun Wu, and Tranzyme Inc. [41]. The derivation and characterization of the HEK293-based Hck-expressing HZ-1 cells have been described elsewhere [19]. HEK239T, HEK293 and HZ1 cells were grown in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Sigma) supplemented with 10% fetal bovine serum (FBS), 0.05 mg/ml penicillin, and 0.05mg/ml streptomycin. HEK293T cells were transfected in 6 well plates using a standard calcium phosphate method. Lentiviral transduction was employed to generate Jurkat cells stably expressing human CD4. Briefly, HEK293 cells were co-transfected with 2.5 μg pDelta8.9, 1.5 μg VSV-G and 3 μg pWPI-puro plasmid containing human CD4 cDNA in Opti-MEM medium with 12 μg PEI. After 5 hours, medium was refreshed with cell culture media. Supernatant was collected 48 hours post-transfection, filtered and used to infect Jurkat cells. Infected cells were selected with 6 μg/ml puromycin for 2 days. Single cell clones of CD4 transduced Jurkat cells were isolated by cellenONE X1 system. THP-1, THP-1 derived macrophages, and Jurkat cells were maintained in RPMI-1640 medium (Sigma) supplemented with 10% FBS, 0.05 mg/ml penicillin, and 0.05 mg/ml streptomycin.

Plasmids

Nef alleles from HIV-1 M SF2 (P03407), HIV-1 N 2693BA (GQ925928), HIV-1 N S4858 (KY498771), HIV-1 N YBF30 (AJ006022), HIV-1 N YBF106 (AJ271370), HIV-1 Letasoft Sound Booster 1.1.88 Free Download with Crack DJO0131 (AY532635), HIV-1 O 13127k4 (AY536904), HIV-1 P RBF168 (GU111555), SIVcpz(P.t.t) CAM5 (AJ271369), SIVcpz(P.t.t) EK505 (DQ373065), SIVcpz(P.t.t) MB897 (EF535994), SIVcpz(P.t.s) ch-Nok5 (AY536915) and SIVcpz(P.t.s) Tan2 (EF394357) were cloned into the expression vector pEBB containing a C-terminal Myc-tag. Human Hck p59 cDNA (isoform b/NP_001165604) was cloned into the pEBB vector together with a 123 aa biotin acceptor domain fused to their C-termini [21]. Hck variants A1, B6, and C3 [25] were created to the same vector background. The Nef mutants (HIV-1 M SF2 Y120F, Y120I, A83E, A83E/Y120I, A83I, A83I/Y120I, A83L, A83L/Y120I, A83M, A83M/Y120I, A83Q, A83Q/Y120I, A83S, A83S/Y120I; HIV-1 N YBF30 Q83M, YBF30 I120F, 2693BA M83Q; SIVcpz(P.t.t) Cam5 I120Y, EK505 E83M, EK505 I120Y and SIVcpz (P.t.s) ch-Nok5 I83Q, ch-Nok5 I120F) were generated in the same vector backbone using standard PCR-assisted mutagenesis. All of the Nef variants mentioned above were cloned into the pWPI-GFP vector (Addgene # 12254) for lentiviral transduction of stably CD4-expressing Jurkat cells and THP-1-derived macrophages.

Immunoblots

Cells were collected and lysed on ice for 10 minutes in lysis buffer (150 mM NaCl, 50 mM Tris-HCl [pH 7.4], 1% NP-40) with protease and phosphatase inhibitors (Thermo Fisher Scientific). Cell lysates were centrifuged at 16,000 x g at 4°C for 5 min. Proteins from cell extracts were analyzed by standard SDS gel electrophoresis and Western blotting using IRDye-labeled detection reagents detailed above.

AP-1 luciferase reporter assay

HZ1 cells were transfected using TransIT-2020 reagent (Mirus) with 50 ng of Nef expression vector together with 50 ng of AP-1 pfLUC reporter plasmid [42] driving the AP-1 inducible expression of firefly luciferase, plus 50 ng of the plasmid pRL-TK (Promega) expressing low and constitutive levels of Renilla luciferase. Cells were collected and lysed with lysis buffer (Promega) on ice. A dual-luciferase reporter assay system from Promega was utilized to determine luciferase activities following the manufacturer’s protocol using Berthold Sirius single-tube luminometer detection.

Lentiviral transduction of THP-1 derived macrophages

1 x 108 THP-1 cells were infected with lentiviral vectors containing various nef Razer Cortex Game Booster 9.14 Crack For Windows Free. 3 days post-infection, GFP positive THP-1 cells were sorted by fluorescence-activated cell sorting (FACS). Subsequently, 1 x 107 sorted THP-1 cells were seeded into each well in a 6-well plate and treated with 10 ng/ml of PMA for 2 days. Adherent cells (M0 macrophages) were further cultured for 2 days in the presence of 10 ng/ml PMA and 10 ng/mL of granulocyte-macrophage colony-stimulating factor (GM-CSF) (ThermoFisher) to differentiate them towards an M1 macrophage phenotype.

Flow cytometry analysis of CD4 downregulation

Nef-expressing HIV-1-like pseudoviruses were produced as described previously [19]. Jurkat cells stably expressing CD4 were infected with such lentiviral vectors containing various nef alleles. 3 days post-infection the cells were collected and washed twice with PBS (pH 7.4), followed by fixing with 1% Paraformaldehyde (Sigma) at room temperature for 20 min. Cells were washed twice with PBS containing 2% FBS (FACS buffer) and stained with Alexa Fluor 647 conjugated anti human CD4 antibody at room temperature for 40 min. After staining the cells were washed twice with FACS buffer and re-suspended in PBS. The CD4 cell surface expression was analyzed using a BD Accuri C6 flow cytometer (BD Biosciences) and data analysis was performed using FlowJo software (version 10.4, Ashland OR: Becton, Dickinson and Company).

Virion infectivity

To determine the effects of Nef on virion infectivity, HEK293T cells were co-transfected with pEBB expression plasmids for different Nefs or an empty vector control and an HIV-1 reporter virus lacking functional nef and vpu genes (HIV-1 NL4-3 Δnef Δvpu IRES eGFP). Two days post transfection, cell culture supernatants were harvested. To quantify infectious virus yield, 6,000 TZM-bl cells were seeded in 96-well plates and infected with the cell culture supernatant of transfected HEK293T cells in triplicate on the following day. 3 days post infection, β-galactosidase reporter gene expression was determined using the GalScreen kit (Applied Bioscience) according to the manufacturer’s instructions. In parallel, p24 concentration was determined using a home-made ELISA. Relative virion infectivity was subsequently calculated by normalizing infectious virus yield to the amount of p24.

Phylogenetic analyses

A maximum-likelihood phylogenetic tree was constructed as described previously [43]. Briefly, the 65 nucleotide sequences of the nef gene included in the analysis were aligned using MUSCLE and the phylogenetic tree was constructed using MEGA7 [44]. Most recent common ancestors (MRCA) of Nef proteins of HIV-1 groups M, O, and N had been inferred in a previous study [33].

Supporting information

S4 Fig. One-hundred ns all-atom molecular dynamics simulations show that stabilization of R77 side chain provided by the R-clamp in the wild-type complex could be reproduced by Met, Ile and Trp in the A83M/Y120I double mutant.

Analysis of the MD trajectories show that in the wild type complex the stacked Tyr-Arg-Trp π-cation-π interaction remains stable, as interpreted from Arg to Tyr and Arg to Trp side chain distances, which show little fluctuations around their average values, 4.2 Å (A) and 3.5 Å (B). In (C) is shown a snapshot from the wild type complex simulation in which these distances are close to their average values. In the double mutant the Arg to Trp distance is on the average about 0.5 Å longer than that in the wild type complex (E), but also remains stable around its average value, 4.0 Å. If 6 Å is taken as the maximum distance for a cation-π interaction [47] the observed distances are well within the limit. Likewise, hydrophobic contacts to and between Met and Ile on the other side of the Arg plane remain relatively stable. Non-bonded contacts are likely to be bolstered by Met sulfur [48]. Ile and Arg sidechains are on average 4.4 Å apart (D). Met to Arg (F) and Ile to Met (G) distances show more variation, but are for the majority of time close to about 4.6 and 3.9 Å. In (H) is shown a snapshot from the double mutant complex simulation in which these distances are close to their average values. A salt bridge between Arg and Asp side chains is present in 96% (WT) and 93% (double mutant) of the simulation frames. MD simulations in explicit solvent were performed with AMBER 20 [24] using the ff14SB force field. The WT and A83M/Y120I complexes were placed in a cubic box with a minimum solute-box distance of 10 Å, and solvated with TIP3P water molecules. Six sodium ions were added to neutralize the system. After minimization, heating and equilibration of the system, the production 100-ns MD simulations were performed WinRAR Free Activate periodic boundary conditions at 300 K. The temperature was maintained by using the Langevin thermostat, whereas the pressure was kept at 1 bar using the Berendsen barostat [49]. The time step was set to 2 fs. Long-range electrostatic interactions were treated using the Particle Mesh Ewald method [50] with a cut-off of 10 Å. Bond lengths involving hydrogen atoms were constrained by SHAKE [51]. Analyses of the trajectories were carried out with CPPTRAJ [52].

https://doi.org/10.1371/journal.ppat.1009728.s004

(PDF)

S5 Fig. Role of R-clamp residues 83/120 in intracellular Hck-Nef complex formation.

HEK293 cells were transfected with Myc-tagged wild-type HIV-1 M SF2 Nef (WT) or its mutants including Y120I, A83L-Y120I, P76A+P78A (AxxA), A83Q, and A83Q-Y120I) together with biotin acceptor domain-tagged SH3 mutated Hck-p59 variants Hck-A1 (A.) or Hck-B6 (B.). Similarly, YBF30 or its R-clamp gain-of-function mutant Q83M were co-expressed with biotin acceptor domain-tagged wild-type Hck-p59 or its SH3 mutant Hck-B6. (C.). The Nef-contacting RT-loop residues centered around the critical Hck SH3 amino acid 13 (indicated in bold) are shown on top of these Figs. Lysates of the transfected cells were subjected to anti-Myc immunoprecipitation followed by Western blotting analysis of the immune complexes using labeled streptavidin (top panels). Equal Hck and Nef expression in the total lysates was confirmed by labeled streptavidin (Hck) (middle panels) or an anti-Myc (Nef) antibody (bottom panels).

https://doi.org/10.1371/journal.ppat.1009728.s005

(PDF)

S6 Fig. Role of the R-clamp in Nef-mediated enhancement of virion infectivity.

(A) HEK293T cells were co-transfected with pEBB expression plasmids for the indicated Nefs and an HIV-1 reporter virus lacking functional nef and vpu genes (HIV-1 NL4-3 Δnef Δvpu IRES eGFP). Two days post transfection cell culture supernatants were harvested. Infectious virus yield was determined by infecting TZM-bl reporter cells and normalized to the amount of p24 (as determined by ELISA) to calculate virion infectivity. Mean values +/- SEM of four independent experiments are shown. (B) Two days post transfection cells were harvested for Western Blot analysis. Nef was detected via an anti-myc tag antibody. GAPDH served as loading control.

https://doi.org/10.1371/journal.ppat.1009728.s006

(PDF)

Acknowledgments

We thank Ms. Virpi Syvälahti for expert technical assistance.

References

  1. 1. Sauter D, Kirchhoff F. Key viral adaptations preceding the AIDS pandemic. Cell Host Microbe. 2019;25(1):27–38. Epub 2019/01/11. pmid:30629915.
  2. 2. Désiré N, Cerutti L, Le Hingrat Q, Perrier M, Emler S, Calvez V, et al. Characterization update of HIV-1 M subtypes diversity and proposal for subtypes A and D sub-subtypes reclassification. Retrovirology. 2018;15(1):80. Epub 2018/12/24. pmid:30577842.
  3. 3. Robertson DL, Anderson JP, Bradac JA, Carr JK, Foley B, Funkhouser RK, et al. HIV-1 nomenclature proposal. Science. 2000;288(5463):55–6. Epub 2000/04/15. pmid:10766634.
  4. 4. Yamaguchi J, Vallari A, McArthur C, Sthreshley L, Cloherty GA, Berg MG, et al. Brief Report: Complete Genome Sequence of CG-0018a-01 Establishes HIV-1 Subtype L. J Acquir Immune Defic Syndr. 2020;83(3):319–22. Epub 2019/11/07. pmid:31693506.
  5. 5. Kirchhoff F, Schindler M, Specht A, Arhel N, Munch J. Role of Nef in primate lentiviral immunopathogenesis. Cell Mol Life Sci. 2008;65(17):2621–36. Epub 2008/04/29. pmid:18438604.
  6. 6. Arhel NJ, Kirchhoff F. Implications of Nef: host cell interactions in viral persistence and progression to AIDS. Curr Top Microbiol Immunol. 2009;339:147–75. Epub 2009/12/17. pmid:20012528.
  7. 7. Saksela K. Interactions of the HIV/SIV pathogenicity factor Nef with SH3 domain-containing host cell proteins. Curr HIV Res. 2011;9(7):531–42. Epub 2011/11/23. pmid:22103837.
  8. 8. Staudt RP, Alvarado JJ, Emert-Sedlak LA, Shi H, Shu ST, Wales TE, et al. Structure, function, and inhibitor targeting of HIV-1 Nef-effector kinase complexes. J Biol Chem. 2020;295(44):15158–71. Epub 2020/08/31. pmid:32862141.
  9. 9. Lee CH, Saksela K, Mirza UA, Gridinsoft Anti-Malware 4.2.5 Crack + Activation Code Free {2021} BT, Kuriyan J. Crystal structure of the conserved core of HIV-1 Nef complexed with a Src family SH3 domain. Cell. 1996;85(6):931–42. Epub 1996/06/14. pmid:8681387.
  10. 10. Moarefi I, LaFevre-Bernt M, Sicheri F, Huse M, Lee CH, Kuriyan J, et al. Activation of the Src-family tyrosine kinase Hck by SH3 domain displacement. Nature. 1997;385(6617):650–3. Epub 1997/02/13. pmid:9024665.
  11. 11. Guiet R, Poincloux R, Castandet J, Marois L, Labrousse A, Le Cabec V, et al. Hematopoietic cell kinase (Hck) isoforms and phagocyte duties—from signaling and actin reorganization to migration and phagocytosis. Eur J Cell Biol. 2008;87(8–9):527–42. Epub 2008/06/10. pmid:18538446.
  12. 12. Kumar A, Herbein G. The macrophage: a therapeutic target in HIV-1 infection. Mol Cell Ther. 2014;2:10. Epub 2014/01/01. pmid:26056579.
  13. 13. Greenberg ME, Iafrate AJ, Skowronski J. The SH3 domain-binding surface and an acidic motif in HIV-1 Nef regulate trafficking of class I MHC complexes. Embo j. 1998;17(10):2777–89. Epub 1998/06/10. pmid:9582271.
  14. 14. Imle A, Abraham L, Tsopoulidis N, Hoflack B, Saksela K, Fackler OT. Association with PAK2 Enables Functional Interactions of Lentiviral Nef Proteins with the Exocyst Complex. MBio. 2015;6(5):e01309–15. pmid:26350970.
  15. 15. Rudolph JM, Eickel N, Haller C, Schindler M, Fackler OT. Inhibition of T-cell receptor-induced actin remodeling and relocalization of Lck are evolutionarily conserved activities of lentiviral Nef proteins. J Virol. 2009;83(22):11528–39. Epub 2009/09/04. pmid:19726522.
  16. 16. Saksela K, Cheng G, Baltimore D. Proline-rich (PxxP) motifs in HIV-1 Nef bind to SH3 domains of a subset of Src kinases and are required for the enhanced growth of Nef+ viruses but not for down-regulation of CD4. EMBO J. 1995;14(3):484–91. Epub 1995/02/01. pmid:7859737.
  17. 17. Mayer BJ. The discovery of modular binding domains: building blocks of cell signalling. Nat Rev Mol Cell Biol. 2015;16(11):691–8. Epub 2015/10/01. pmid:26420231.
  18. 18. Saksela K, Permi P. SH3 domain ligand binding: What’s the consensus and where’s the specificity? FEBS Lett. 2012;586(17):2609–14. Epub 2012/06/20. pmid:22710157.
  19. 19. Zhao Z, Fagerlund R, Baur AS, Saksela K. HIV-1 Nef-induced secretion of the proinflammatory protease TACE into extracellularvesicles is mediated by Raf-1, and can be suppressed by clinical protein kinase inhibitors. J Virol. 2021. Epub 2021/02/19. pmid:33597213.
  20. 20. Collette Y, Arold S, Picard C, Janvier K, Benichou S, Benarous R, et al. HIV-2 and SIV nef proteins target different Src family SH3 domains than does HIV-1 Nef because of a triple amino acid substitution. J Biol Chem. 2000;275(6):4171–6. Epub 2000/02/08. pmid:10660579.
  21. 21. Zhao Z, Kesti T, Ugurlu H, Baur AS, Fagerlund R, Saksela K. Tyrosine phosphorylation directs TACE into extracellular vesicles via unconventional secretion. Traffic. 2019;20(3):202–12. Epub 2018/12/21. pmid:30569492.
  22. 22. Kirchhoff F, Schindler M, Bailer N, Renkema GH, Saksela K, Knoop V, et al. Nef proteins from simian immunodeficiency virus-infected chimpanzees interact with p21-activated kinase 2 and modulate cell surface expression of various human receptors. J Virol. 2004;78(13):6864–74. Epub 2004/06/15. pmid:15194762.
  23. 23. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, et al. UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem. 2004;25(13):1605–12. Epub 2004/07/21. pmid:15264254.
  24. 24. Case DA, Belfon K, Ben-Shalom IY, Brozell SR, Cerutti DS, Cheatham TEI, et al. AMBER 2020: University of California, San Francisco; 2020.
  25. 25. Hiipakka M, Poikonen K, Saksela K. SH3 domains with high affinity and engineered ligand specificities targeted to HIV-1 Nef. J Mol Biol. 1999;293(5):1097–106. Epub 1999/11/05. pmid:10547288.
  26. 26. Horenkamp FA, Breuer S, Schulte A, Lulf S, Weyand M, Saksela K, et al. Conformation of the dileucine-based sorting motif in HIV-1 Nef revealed by intermolecular domain assembly. Traffic. 2011;12(7):867–77. Epub 2011/04/12. pmid:21477083.
  27. 27. Chowers MY, Spina CA, Kwoh TJ, Fitch NJ, Richman DD, Guatelli JC. Optimal infectivity in vitro of human immunodeficiency virus type 1 requires an intact nef gene. J Virol. 1994;68(5):2906–14. Epub 1994/05/01. pmid:8151761.
  28. 28. Firrito C, Bertelli C, Vanzo T, Chande A, Pizzato M. SERINC5 as a New Restriction Factor for Human Immunodeficiency Virus and Murine Leukemia Virus. Annu Rev Virol. 2018;5(1):323–40. Epub 2018/09/29. pmid:30265629.
  29. 29. Shi J, Xiong R, Zhou T, Su P, Zhang X, Qiu X, et al. HIV-1 Nef antagonizes SERINC5 restriction by downregulation of SERINC5 via the endosome/lysosome system. J Virol. 2018;92(11). Epub 2018/03/09. pmid:29514909.
  30. 30. Fackler OT, Moris A, Tibroni N, Giese SI, Glass B, Schwartz O, et al. Functional characterization of HIV-1 Nef mutants in the context of viral infection. Virology. 2006;351(2):322–39. Epub 2006/05/11. pmid:16684552.
  31. 31. Collette Y, Dutartre H, Benziane A, Ramos M, Benarous R, Harris M, et al. Physical and functional interaction of Nef with Lck. HIV-1 Nef-induced T-cell signaling defects. J Biol Chem. 1996;271(11):6333–41. Epub 1996/03/15. pmid:8626429.
  32. 32. Rauch S, Pulkkinen K, Saksela K, Fackler OT. Human immunodeficiency virus type 1 Nef recruits the guanine exchange factor Vav1 via an unexpected interface into plasma membrane microdomains for association with p21-activated kinase 2 activity. J Virol. 2008;82(6):2918–29. Epub 2007/12/21. pmid:18094167.
  33. 33. Blagoveshchenskaya AD, Thomas L, Feliciangeli SF, Hung CH, Thomas G. HIV-1 Nef downregulates MHC-I by a PACS-1- and PI3K-regulated ARF6 endocytic pathway. Cell. 2002;111(6):853–66. Epub 2003/01/16. pmid:12526811.
  34. 34. Tarafdar S, Poe JA, Smithgall TE. The accessory factor Nef links HIV-1 to Tec/Btk kinases in an Src homology 3 domain-dependent manner. J Biol Chem. 2014;289(22):15718–28. Epub 2014/04/12. pmid:24722985.
  35. 35. Kluge SF, Mack K, Iyer SS, Pujol FM, Heigele A, Learn GH, et al. Nef proteins of epidemic HIV-1 group O strains antagonize human tetherin. Cell Host Microbe. 2014;16(5):639–50. Epub 2014/12/20. pmid:25525794.
  36. 36. Bibollet-Ruche F, Heigele A, Keele BF, Easlick JL, Decker JM, Takehisa J, et al. Efficient SIVcpz replication in human lymphoid tissue requires viral matrix protein adaptation. J Clin Invest. 2012;122(5):1644–52. Epub 2012/04/17. pmid:22505456.
  37. 37. Sauter D, Hué S, Petit SJ, Plantier JC, Towers GJ, Kirchhoff F, et al. HIV-1 Group P is unable to antagonize human tetherin by Vpu, Env or Nef. Retrovirology. 2011;8:103. Epub 2011/12/17. pmid:22171785.
  38. 38. Wertheim JO, Worobey M. Dating the age of the SIV lineages that gave rise to HIV-1 and HIV-2. PLoS Comput Biol. 2009;5(5):e1000377. Epub 2009/05/05. pmid:19412344.
  39. 39. Delaugerre C, De Oliveira F, Lascoux-Combe C, Plantier JC, Simon F. HIV-1 group N: travelling beyond Cameroon. Lancet. 2011;378(9806):1894. Epub 2011/11/29. pmid:22118443.
  40. 40. Srinivasan A, York D, Butler Jr., Jannoun-Nasr R, Getchell J, McCormick J, et al. Molecular characterization of HIV-1 isolated from a serum collected in 1976: nucleotide sequence comparison to recent isolates and generation of hybrid HIV. AIDS Res Hum Retroviruses. 1989;5(2):121–9. Epub 1989/04/01. pmid:2713163.
  41. 41. Choi HJ, Smithgall TE. Conserved residues in the HIV-1 Nef hydrophobic pocket are essential for recruitment and activation of the Hck tyrosine kinase. J Mol Biol. 2004;343(5):1255–68. Epub 2004/10/20. acid properties - Crack Key For U.
  42. 42. Aldehaiman A, Momin AA, Restouin A, Wang L, Shi X, Aljedani S, et al. Synergy and allostery in ligand binding by HIV-1 Nef. Biochem J. 2021;478(8):1525–45. Epub 2021/04/01. pmid:33787846.
  43. 43. Platt EJ, Wehrly K, Kuhmann SE, Chesebro B, Kabat D. Effects of CCR5 and CD4 cell surface concentrations on infections by macrophagetropic isolates of human immunodeficiency virus type 1. J Virol. 1998;72(4):2855–64. Epub 1998/04/03. pmid:9525605.
  44. 44. Pesu M, Takaluoma K, Aittomaki S, Lagerstedt A, Saksela K, Kovanen PE, et al. Interleukin-4-induced transcriptional activation by Foobar2000 1.6.7 Beta 10 Crack With Serial Key Free Download 2021 involves multiple serine/threonine kinase pathways and serine phosphorylation of stat6. Blood. 2000;95(2):494–502. Epub 2000/01/11. pmid:10627454.
  45. 45. Nakano Y, Yamamoto K, Ueda MT, Soper A, Konno Y, Kimura I, et al. A role for gorilla APOBEC3G in shaping lentivirus winx dvd ripper platinum speed - Free Activators including transmission to humans. PLoS Pathog. 2020;16(9):e1008812. Epub 2020/09/12. pmid:32913367.
  46. 46. Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016;33(7):1870–4. Epub 2016/03/24. pmid:27004904.
  47. 47. Gallivan JP, Dougherty DA. NinjaGram (Instagram Bot) 7.6.5.3 Crack + Serial Key Free 2021 interactions in structural biology. Proc Natl Acad Sci U S A. 1999;96(17):9459–64. Epub 1999/08/18. pmid:10449714.
  48. 48. Gómez-Tamayo JC, Cordomí A, Olivella M, Mayol E, Fourmy D, Pardo L. Analysis of the interactions of sulfur-containing amino acids in membrane proteins. Protein Sci. 2016;25(8):1517–24. Epub 2016/05/31. pmid:27240306.
  49. 49. Berendsen HJC, Postma JPM, van Gunsteren WF, DiNola A, Haak JR. Molecular dynamics with coupling to an external bath. J Chem Phys. 1984;81:3684–90.
  50. 50. Darden T, York D, Pedersen L. Particle mesh Ewald: an N·log(N) method for Ewald sums in large systems. J Chem Phys. 1993;98:10089–92.
  51. 51. Ryckaert J-P, Ciccotti G, Berendsen HJC. Numerical integration of the cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes. J Comput Phys. 1977;23:327–41.
  52. 52. Roe DR, Cheatham TE 3rd. PTRAJ and CPPTRAJ: Software for Processing and Analysis of Molecular Dynamics Trajectory Data. J Chem Theory Comput. 2013;9(7):3084–95. Epub 2013/07/09. pmid:26583988.
Check for updates via CrossMark

Subject Areas

?

For more information about PLOS Subject Areas, click here.

We want your feedback.Do these Subject Areas make sense for this article? Click the target next to the incorrect Subject Area and let us know. Thanks for your help!

  • HIV-1 
  • Transfection 
  • Viral evolution 
  • Chimpanzees 
  • Macrophages 
  • Primates 
  • SIV 
  • Luciferase 
Источник: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1009728
acid properties  - Crack Key For U

Acid properties - Crack Key For U -

 

Through this website we are seeking historical materials relating to fuel cells. We have constructed the site to gather information from people already familiar with the technology–people such as inventors, researchers, manufacturers, electricians, and marketers. This Basics section presents a general overview of fuel cells for casual visitors.

What is a fuel cell?

A fuel cell is a device that generates electricity by a chemical reaction. Every fuel cell has two electrodes called, respectively, the anode and cathode. The reactions that produce electricity take place at the electrodes.

Every fuel cell also has an electrolyte, which carries electrically charged particles from one electrode to the other, and a catalyst, which speeds the reactions at the electrodes.

Hydrogen is the basic fuel, but fuel cells also require oxygen. One great appeal of fuel cells is that they generate electricity with very little pollution–much of the hydrogen and oxygen used in generating electricity ultimately combine to form a harmless byproduct, namely water.

One detail of terminology: a single fuel cell generates a tiny amount of direct current (DC) electricity. In practice, many fuel cells are usually assembled into a stack. Cell or stack, the principles are the same.

Top

How do fuel cells work?

The purpose of a fuel cell is to produce an electrical current that can be directed outside the cell to do work, such as powering an electric motor or illuminating a light bulb or a city. Because of the way electricity behaves, this current returns to the fuel cell, completing an electrical circuit. (To learn more about electricity and electric power, visit "Throw The Switch" on the Smithsonian website Powering a Generation of Change.) The chemical reactions that produce this current are the key to how a fuel cell works.

There are several kinds of fuel cells, and each operates a bit differently. But in general terms, hydrogen atoms enter a fuel cell at the anode where a chemical reaction strips them of their electrons. The hydrogen atoms are now "ionized," and carry a positive electrical charge. The negatively charged electrons provide the current through wires to do work. If alternating current (AC) is needed, the DC output of the fuel cell must be routed through a conversion device called an inverter.

animated image showing the function of a PEM  fuel cell
Graphic by Marc Marshall, Schatz Energy Research Center

Oxygen enters the fuel cell at the cathode and, in some cell types (like the one illustrated above), it there combines with electrons returning from the electrical circuit and hydrogen ions that have traveled through the electrolyte from the anode. In other cell types the oxygen picks up electrons and then travels through the electrolyte to the anode, where it combines with hydrogen ions.

The electrolyte plays a key role. It must permit only the appropriate ions to pass between the anode and cathode. If free electrons or other substances could travel through the electrolyte, they would disrupt the chemical reaction.

Whether they combine at anode or cathode, together hydrogen and oxygen form water, which drains from the cell. As long as a fuel cell is supplied with hydrogen and oxygen, it will generate electricity.

Even better, since fuel cells create electricity chemically, rather than by combustion, they are not subject to the thermodynamic laws that limit a conventional power plant (see "Carnot Limit" in the glossary). Therefore, fuel cells are more efficient in extracting energy from a fuel. Waste heat from some cells can also be harnessed, boosting system efficiency still further.

Top

So why can't I go out and buy a fuel cell?

The basic workings of a fuel cell may not be difficult to illustrate. But building inexpensive, efficient, reliable fuel cells is a far more complicated business.

Scientists and inventors have designed many different types and sizes of fuel cells in the search for greater efficiency, and the technical details of each kind vary. Many of the choices facing fuel cell developers are constrained by the choice of electrolyte. The design of electrodes, for example, and the materials used to make them depend on the electrolyte. Today, the main electrolyte types are alkali, molten carbonate, phosphoric acid, proton exchange membrane (PEM) and solid oxide. The first three are liquid electrolytes; the last two are solids.

The type of fuel also depends on the electrolyte. Some cells need pure hydrogen, and therefore demand extra equipment such as a "reformer" to purify the fuel. Other cells can tolerate some impurities, but might need higher temperatures to run efficiently. Liquid electrolytes circulate in some cells, which requires pumps. The type of electrolyte also dictates a cell's operating temperature–"molten" carbonate cells run hot, just as the name implies.

Each type of fuel cell has advantages and drawbacks compared to the others, and none is yet cheap and efficient enough to widely replace traditional ways of generating power, such coal-fired, hydroelectric, or even nuclear power plants.

The following list describes the five main types of fuel cells. More detailed information can be found in those specific areas of this site.

Top

Different types of fuel cells.

drawing of an Alkali fuel cell
Drawing of an alkali cell.
Alkali fuel cells operate on compressed hydrogen and oxygen. They generally use a solution of potassium hydroxide (chemically, KOH) in water as their electrolyte. Efficiency is about 70 percent, and operating temperature is 150 to 200 degrees C, (about 300 to 400 degrees F). Cell output ranges from 300 watts (W) to 5 kilowatts (kW). Alkali cells were used in Apollo spacecraft to provide both electricity and drinking water. They require pure hydrogen fuel, however, and their platinum electrode catalysts are expensive. And like any container filled with liquid, they can leak.
drawing of molten carbonate fuel cell
Drawing of a molten carbonate cell
Molten Carbonate fuel cells (MCFC) use high-temperature compounds of salt (like sodium or magnesium) carbonates (chemically, CO3) as the electrolyte. Efficiency ranges from 60 to 80 percent, and operating temperature is about 650 degrees C (1,200 degrees F). Units with output up to 2 megawatts (MW) have been constructed, and designs exist for units up to 100 MW. The high temperature limits damage from carbon monoxide "poisoning" of the cell and waste heat can be recycled to make additional electricity. Their nickel electrode-catalysts are inexpensive compared to the platinum used in other cells. But the high temperature also limits the materials and safe uses of MCFCs–they would probably be too hot for home use. Also, carbonate ions from the electrolyte are used up in the reactions, making it necessary to inject carbon dioxide to compensate.

Phosphoric Acid fuel cells (PAFC) use phosphoric acid as the electrolyte. Efficiency ranges from 40 to 80 percent, and operating temperature is between 150 to 200 degrees C (about 300 to 400 degrees F). Existing phosphoric acid cells have outputs up to 200 kW, and 11 MW units have been tested. PAFCs tolerate a carbon monoxide concentration of about 1.5 percent, which broadens the choice of fuels they can use. If gasoline is used, the sulfur must be removed. Platinum electrode-catalysts are needed, and internal parts must be able to withstand the corrosive acid.

drawing of how both phosphoric acid and PEM fuel cells operate
Drawing of how both phosphoric acid and PEM fuel cells operate.

Proton Exchange Membrane (PEM) fuel cells work with a polymer electrolyte in the form of a thin, permeable sheet. Efficiency is about 40 to 50 percent, and operating temperature is about 80 degrees C (about 175 degrees F). Cell outputs generally range from 50 to 250 kW. The solid, flexible electrolyte will not leak or crack, and these cells operate at a low enough temperature to make them suitable for homes and cars. But their fuels must be purified, and a platinum catalyst is used on both sides of the membrane, raising costs.

drawing of solid oxide fuel cell
Drawing of a solid oxide cell
Solid Oxide fuel cells (SOFC) use a hard, ceramic compound of metal (like calcium or zirconium) oxides (chemically, O2) as electrolyte. Efficiency is about 60 percent, and operating temperatures are about 1,000 degrees C (about 1,800 degrees F). Cells output is up to 100 kW. At such high temperatures a reformer is not required to extract hydrogen from the fuel, and waste heat can be recycled to make additional electricity. However, the high temperature limits applications of SOFC units and they tend to be rather large. While solid electrolytes cannot leak, they can crack.

More detailed information about each fuel cell type, including histories and current applications, can be found on their specific parts of this site. We have also provided a glossary of technical terms–a link is provided at the top of each technology page.

Top

©2017 Smithsonian Institution
(Copyright Statement)
 

 
Источник: https://americanhistory.si.edu/fuelcells/basics.htm

Acid Rain

Inorganic Reactions Experiment

Authors: Rachel Casiday and Regina Frey
Department of Chemistry, Washington University
St. Louis, MO 63130


Natural Acidity of Rainwater

Pure water has a pH of 7.0 (neutral); however, natural, unpolluted rainwater actually has a pH of about 5.6 (acidic).[Recall from Experiment 1 that pH is a measure of the hydrogen ion (H+) concentration.] The acidity of rainwater comes from the natural presence of three substances (CO2, NO, and SO2) found in the troposphere (the lowest layer of the atmosphere). As is seen in Table I, carbon dioxide (CO2) is present in the greatest concentration and therefore contributes the most to the natural acidity of rainwater.

Gas

Natural Sources

Concentration

Carbon dioxide
CO2
Decomposition 355 ppm
Nitric oxide
NO
Electric discharge 0.01 ppm
Sulfur dioxide
SO2
Volcanic gases 0-0.01 ppm

Table 1

Carbon dioxide, produced in the decomposition of organic material, is the primary source of acidity in unpolluted rainwater.

NOTE: Parts per million (ppm) is a common concentration measure used in environmental chemistry. The formula for ppm is given by:

Carbon dioxide reacts with water to form carbonic acid (Equation 1). Carbonic acid then dissociates to give the hydrogen ion (H+) and the hydrogen carbonate ion (HCO3-) (Equation 2). The ability of H2CO3 to deliver H+ is what classifies this molecule as an acid, thus lowering the pH of a solution.


(1)

 


(2)

Nitric oxide (NO), which also contributes to the natural acidity of rainwater, is formed during lightning storms by the reaction of nitrogen and oxygen, two common atmospheric gases (Equation 3). In air, NO is oxidized to nitrogen dioxide (NO2) (Equation 4), which in turn reacts with water to give nitric acid (HNO3) (Equation 5). This acid dissociates in water to yield hydrogen ions and nitrate ions (NO3-) in a reaction analagous to the dissociation of carbonic acid shown in Equation 2, again lowering the pH of the solution.


(3)

 


(4)

 


(5)

Acidity of Polluted Rainwater

Unfortunately, human industrial activity produces additional acid-forming compounds in far greater quantities than the natural sources of acidity described above. In some areas of the United States, the pH of rainwater can be 3.0 or lower, approximately 1000 times more acidic than normal rainwater. In 1982, the pH of a fog on the West Coast of the United States was measured at 1.8! When rainwater is too acidic, it can cause problems ranging from killing freshwater fish and damaging crops, to eroding buildings and monuments.


Questions on Acidity of Rainwater

1. List two or more ways that you could test the acidity of a sample of rainwater.

2. Write a balanced chemical equation for the dissociation of nitric acid in water. (HINT: Draw an analogy with Equation 2.)

3. The gaseous oxides found in the atmosphere, including CO2 and NO are nonmetal oxides. What would happen to the pH of rainwater if the atmosphere contained metal oxides instead? (HINT: Think back to Experiment 1.) Briefly, explain your answer.


Sources of Excess Acidity in Rainwater

What causes such a dramatic increase in the acidity of rain relative to pure water? The answer lies within the concentrations of nitric oxide and sulfur dioxide in polluted air. As shown in Table II and Figure 1, the concentrations of these oxides are much higher than in clean air.

Gas

Non-Natural Sources

Concentration

Nitric oxide
NO
Internal Combustion0.2 ppm
Sulfur dioxide
SO2
Fossil-fuel Combustion 0.1 - 2.0 ppm

Table II

Humans cause many combustion processes that dramatically increase the concentrations of acid-producing oxides in the atmosphere. Although CO2 is present in a much higher concentration than NO and SO2, CO2 does not form acid to the same extent as the other two gases. Thus, a large increase in the concentration of NO and SO2 significantly affects the pH of rainwater, even though both gases are present at much lower concentration than CO2.

Figure 1

Comparison of the concentrations of NO and SO2 in clean and polluted air.

About one-fourth of the acidity of rain is accounted for by nitric acid (HNO3). In addition to the natural processes that form small amounts of nitric acid in rainwater, high-temperature air combustion, such as occurs in car engines and power plants, produces large amounts of NO gas. This gas then forms nitric acid via Equations 4 and 5. Thus, a process that occurs naturally at levels tolerable by the environment can harm the environment when human activity causes the process (e.g., formation of nitric acid) to occur to a much greater extent.

What about the other 75% of the acidity of rain? Most is accounted for by the presence of sulfuric acid (H2SO4) in rainwater. Although sulfuric acid may be produced naturally in small quantities from biological decay and volcanic activity (Figure 1), it is produced almost entirely by human activity, especially the combustion of sulfur-containing fossil fuels in power plants. When these fossil fuels are burned, the sulfur contained in them reacts with oxygen from the air to form sulfur dioxide (SO2). Combustion of fossil fuels accounts for approximately 80% of the total atmospheric SO2 in the United States. The effects of burning fossil fuels can be dramatic: in contrast to the unpolluted atmospheric SO2 concentration of 0 to 0.01 ppm, polluted urban air can contain 0.1 to 2 ppm SO2, or up to 200 times more SO2! Sulfur dioxide, like the oxides of carbon and nitrogen, reacts with water to form sulfuric acid (Equation 6).


(6)

Sulfuric acid is a strong acid, so it readily dissociates in water, to give an H+ ion and an HSO4- ion (Equation 7). The HSO4- ion may further dissociate to give H+ and SO42- (Equation 8). Thus, the presence of H2SO4 causes the concentration of H+ ions to increase dramatically, and so the pH of the rainwater drops to harmful levels.


(7)

 


(8)


Questions on Sources of Acidity in Rainwater

4. At sea level and 25oC, one mole of air fills a volume of 24.5 liters, and the density of air is 1.22x10-6 g/ml. Compute the mole fraction (i.e.,moles of component /total moles) and molarity of SO2 when the atmospheric concentration of SO2 is 2.0 ppm (see note in Table I).

5.One strategy for limiting the amount of acid pollution in the atmosphere is scrubbing. In particular, calcium oxide (CaO) is injected into the combustion chamber of a power plant, where it reacts with the sulfur dioxide produced, to yield solid calcium sulfite.

a. Write a balanced chemical equation for this reaction. (HINT: Consult the table of common ions in the tutorial assignment for Experiment 1 to view the structure and formula for sulfite; also, use your knowledge of the periodic table to deduce the charge of the calcium ion. Using these facts, you can deduce the formula for calcium sulfite.)

b. Approximately one ton, or 9.0x102 kg, of calcium sulfite is generated each year for every person served by a power plant. How much sulfur dioxide (in moles) is prevented from entering the atmosphere when this much calcium sulfite is generated? Show your calculation.

c. The final stage in the scrubbing process is to treat the combustion gases with a slurry of solid CaO in water, in order to trap any remaining SO2 and convert it to calcium sulfite. A slurry is a thick suspension of an insoluble precipitate in water. Using the solubility guidelines provided in the lab manual for this experiment, predict whether this stage of the scrubbing process will produce a slurry (i.e., precipitate) or a solution (i.e., no precipitate) of calcium sulfite .

d. If MgO, rather than CaO, were used for scrubbing, would the product of the final stage be a slurry or a solution of magnesium sulfite? (Assume that a very large quantity of magnesium sulfite, relative to the amount of water, is produced.)


Environmental Effects of Acid Rain

Acid rain triggers a number of inorganic and biochemical reactions with deleterious environmental effects, making this a growing environmental problem worldwide.

  • Many lakes have become so acidic that fish cannot live in them anymore.
  • Degradation of many soil minerals produces metal ions that are then washed away in the runoff, causing several effects:
    • The release of toxic ions, such as Al3+, into the water supply.
    • The loss of important minerals, such as Ca2+, from the soil, killing trees and damaging crops.
  • Atmospheric pollutants are easily moved by wind currents, so acid-rain effects are felt far from where pollutants are generated.

Stone Buildings and Monuments in Acid Rain

Marble and limestone have long been preferred materials for constructing durable buildings and monuments. The Saint Louis Art Museum, the Parthenon in Greece, the Chicago Field Museum, and the United States Capitol building are all made of these materials. Marble and limestone both consist of calcium carbonate (CaCO3), and differ only in their crystalline structure. Limestone consists of smaller crystals and is more porous than marble; it is used more extensively in buildings. Marble, with its larger crystals and smaller pores, can attain a high polish and is thus preferred for monuments and statues. Although these are recognized as highly durable materials, buildings and outdoor monuments made of marble and limestone are now being gradually eroded away by acid rain.

How does this happen? A chemical reaction (Equation 9) between calcium carbonate and sulfuric acid (the primary acid component of acid rain) results in the dissolution of CaCO3 to give aqueous ions, which in turn are washed away in the water flow.


(9)

This process occurs at the surface of the buildings or monuments; thus acid rain can easily destroy the details on relief work (e.g., the faces on a statue), but generally does not affect the structural integrity of the building. The degree of damage is determined not only by the acidity of the rainwater, but also by the amount of water flow that a region of the surface receives. Regions exposed to direct downpour of acid rain are highly susceptible to erosion, but regions that are more sheltered from water flow (such as under eaves and overhangs of limestone buildings) are much better preserved. The marble columns of the emperors Marcus Aurelius and Trajan, in Rome, provide a striking example: large volumes of rainwater flow directly over certain parts of the columns, which have been badly eroded; other parts are protected by wind effects from this flow, and are in extremely good condition even after nearly 2000 years!

Even those parts of marble and limestone structures that are not themselves eroded can be damaged by this process (Equation 9). When the water dries, it leaves behind the ions that were dissolved in it. When a solution containing calcium and sulfate ions dries, the ions crystallize as CaSO4l 2H2O, which is gypsum. Gypsum is soluble in water, so it is washed away from areas that receive a heavy flow of rain. However, gypsum accumulates in the same sheltered areas that are protected from erosion, and attracts dust, carbon particles, dry-ash, and other dark pollutants. This results in blackening of the surfaces where gypsum accumulates.

An even more serious situation arises when water containing calcium and sulfate ions penetrates the stone's pores. When the water dries, the ions form salt crystals within the pore system. These crystals can disrupt the crystalline arrangement of the atoms in the stone, causing the fundamental structure of the stone to be disturbed. If the crystalline structure is disrupted sufficiently, the stone may actually crack. Thus, porosity is an important factor in determining a stone's durability.


Questions on Effects of Acid Rain

6. Based on the information described above about the calcium ion, and the formula of calcium carbonate (CaCO3), deduce the charge of the carbonate ion. Also, in the structure of the carbonate ion, are any of the oxygens bonded to one another, or all the oxygens bonded to the carbon atom? (HINT: Consult the structure of the common ions given in the tutorial for Experiment 1).

7. In water, H2SO4 can dissociate to yield two H+ ions and one SO42- ion. Write the net ionic equation for the reaction of calcium carbonate and sulfuric acid. (See the introduction to Experiment 2 in the lab manual for a discussion of net ionic equations.)

8. Which is a more durable building material, limestone or marble? Briefly, explain your reasoning.

Additional Links:


References:

Brown, Lemay, and Buster. Chemistry: the Central Science, 7th ed. Upper Saddle River, NJ: Prentice Hall, 1997. p. 673-5.

Charola, A. "Acid Rain Effects on Stone Monuments," J. Chem. Ed.64 (1987), p. 436-7.

Petrucci and Harwood. General Chemistry: Principles and Modern Applications, 7th ed. Upper Saddle River, NJ: Prentice Hall, 1997. p. 614-5.

Walk, M. F. and P.J. Godfrey. "Effects of Acid Deposition on Surface Waters," J. New England Water Works Assn. Dec. 1990, p. 248-251.

Zumdahl, S.. Chem. Principles, 3rd ed. Boston: Houghton Mifflin, 1998. p. 174-6.

Stryer, L. Biochemistry, 4th ed., W.H. Freeman and Co., New York, 1995, p. 332-339.


Acknowledgements:

The authors thank Dewey Holten (Washington University) for many helpful suggestions in the writing of this tutorial.

The development of this tutorial was supported by a grant from the Howard Hughes Medical Institute, through the Undergraduate Biological Sciences Education program, Grant HHMI# 71192-502004 to Washington University.

Copyright 1998, Washington University, All Rights Reserved.

Источник: http://www.chemistry.wustl.edu/~edudev/LabTutorials/Water/FreshWater/acidrain.html

How to heal dry, cracked heels, according to dermatologists

A long winter and spring stuck inside in the dry air may have made the skin on your feet, especially your heels, super dry — dry enough to crack like a fault line. And while COVID-related self-isolation may tempt you to keep your feet hidden, cracks in your heels can fracture into deep cuts, or fissures, that can be pretty painful and even get infected. We asked board certified dermatologists Sheel Desai Solomon, MD, founder of Preston Dermatology & Skin Surgery in North Carolina and Samer Jaber, MD, founder of Washington Square Dermatology in New York City, what causes dry, cracked heels and the best treatment for dry heels.

IN THIS ARTICLE What causes dry, cracked heels Quizlet. You should perform the test a few times for each solution. All plants need water, minerals, carbon dioxide, sunlight, and living space. Part One: Flame Tests (As an option, this could be a demo rather than a student activity) When solutions of metals are heated in a Bunsen burner flame, they give off characteristic colours. Electron energy and light lab answer key. 3 Electron Orbitals and electron configurations Electron. Here are some of our favorite virtual lab activities for the classroom. General Chemistry. Learn vocabulary, terms, and more with flashcards, games, and other study tools. Mystery Science. Convert each of the wavelengths in the Data Table from nanometers to meters. pdf Download Selected Download. Turn the collar on the Bunsen burner so that you have an invisible or pale blue flame. What are the alkali metal elements? Where are they located on the periodic table? 2. This test works well for metal ions, and was perfected by Robert Bunsen (1811 – 1899). Orbital Model Activity; Electron. * This simulation take the spectral data from NATIONAL INSTITUTE OF STANDARDS AND TECHNOLOGY. In addition to this, a printable 22 page activity booklet contains student activities. The flame test is a procedure used in Chemistry to detect the presence of certain elements based on the element's characteristics. Kind of a lab safety answer key is the older symbols, always add small amounts of information would be found worksheet will fan the words. 1) Does your reaction have oxygen as one of it's reactants • In a flame test, copper ions emit a deep blue-green light, much more blue than the flame test for barium. analytical calibration curve from flame test lab worksheet answer key , source:terpconnect. Important! Before you view the answer key, decide whether or not you plan to request an extension. To Identify An Unknown Metallic Ion By Means Of Its Flame Test. No two metals emit the exact same color. Place into each test tube 5 drops of the appropriate hydrocarbon. In the video, the scientist will expose seven different salts to a flame. Grasp one presoaked toothpick with forceps. ) Light a wood splint and place inside the test tube. Bookmark File PDF Flame Test Lab 10 Answer Key Flame Test Lab Activity Key flame test. To do a flame test on a metallic element, the metal is first dissolved in a solution and the solution is then held in the hot, blue flame of a Bunsen burner. Flame Test Lab Worksheet Answer Key with 22 Best Chemistry Unit 4 Review Images On Pinterest. Answer Key. Convert each of the wavelengths in the Data Table from nanometers to meters. see results table above. CAUTION: too much air can extinguish the flame. 3 Electron Orbitals and electron configurations Electron. * This simulation take the spectral data from NATIONAL INSTITUTE OF STANDARDS AND TECHNOLOGY. 4 11/20/15 Pre-Lab Questions. Give the product for the following reactions. This test works well for metal ions, and was perfected by Robert Bunsen (1811 – 1899). Pre Lab Flame Tests Lab in your lab notebook Finish Calculations for light properties worksheet for homework if you did not complete it in class. Introduction to the Flame Test Lab: The Flame Test lab was an in-class lab where we tested chemicals in the flames to see the wide range of colors in the color spectrum. Atwood Hydro Flame Furnace - Scholarships. By Compound Interest. July 26, 2017 Flame Test Lab Introduction/Pre-Lab: The purpose of this lab was to observe the effect of certain compounds and more specifically elements in the compounds being burned. The purpose of the flame test lab is to observe the characteristics colors produced by certain metallic Ions when vaporized in a flame. Purpose: To Observe The Characteristic Colors Produced By Metallic Ions When Heated In A Flame. FLAME TEST LAB. If you click on this link , you will be able to find a well crafted sample of a lab report with a short and clear conclusion bearing all the recommended features. Carefully light Bunsen Burner as instructed. Br2, FeBr3 Cl2, FeCl3 b) 2 Post-Lab Questions 1. Be sure to show all work, round answers, and include units on all answers. Bookmark File PDF Flame Test Lab 10 Answer Key Flame Test Lab Activity Key flame test. The potassium flame can be seen in the presence of sodium by viewing the flame through a dark blue cobalt-glass filter, which absorbs the yellow light from Na, but allows the light from K to pass. CAUTION: too much air can extinguish the flame. Important! Before you view the answer key, decide whether or not you plan to request an extension. Flame test with lab write-up; To make-up Flame Test Lab. This test works well for metal ions, and was perfected by Robert Bunsen (1811 – 1899). Analysis Of Cations And Anions Group IV I. You can read Flame Test Lab Answers Key PDF direct on your mobile phones or PC. Lab Electron Configuration of Atoms and Ions Honors Chemistry from flame test lab worksheet answer key , source:yumpu. Flame Test 1. Flame Test Lab Worksheet Answer Key Did You Hear About Math Worksheet Answer Key are a type of education aid. This lab is provided as a hands on way to test an unknown for the identity of cation and anion by using a flame; Question: Module 5 Lab Report - CHEM 1806 Anions, Cations, and Ionic Reactions Follow the directions in the lab write up. This graphic looks at the colour of various metal and metalloid ions that occur during flame tests. Part I Procedure. Convert each of the wavelengths in the Data Table from nanometers to meters. Learn how in 5 minutes with a tutorial resource. Carefully light Bunsen Burner as instructed. Hold the splint in the flame and record the color of the flame that is produced. To do a flame test on a metallic element, the metal is first dissolved in a solution and the solution is then held in the hot, blue flame of a Bunsen burner. The purpose of this experiment was to observe the emission spectrum of atoms in a flame test. The color of the light depends on the frequency of the light given off. This experiment is a classic, must-do experiment in your chemistry class! dichotomous key (6) differentiation (1) diffusion (1) digestive system (1) digital resources (14) disney (1) distance learning (15). Flame Test Post Lab Answer Key. Test Answer Key In this site is not the thesame as a answer encyclopedia you' ' chemistry atomic structure test answer key taftaf de june 20th, 2018 - read and download chemistry atomic structure test answer key free ebooks in pdf format vocabulary for the college bound student answer key online jefferson math '. By measuring the emitted light, we can detect the atoms. Learn vocabulary, terms, and more with flashcards, games, and other study tools. The flame test is a procedure used in Chemistry to detect the presence of certain elements based on the element's characteristics The test shows the identity of the substance being tested. Part I Procedure. Hold the test tube at a slight angle over the candle flame. Flame Test Lab PURPOSE: Using an elements spectrum, learning how the electrons move around in the atom. Organic Chemistry 1 Final Exam Review Study Guide Multiple Choice Test Flame Test Lab; Beers Law Simulation Lab AP Chemistry 1 Textbook: Section 6. Answer Key. Using Worksheets indicates facilitating students to be able to solution questions about matters they have learned. Bookmark File PDF Flame Test Lab 10 Answer Key Flame Test Lab Activity Key flame test. Calculate the quantity of heat, in kilojoules, (a) required to raise the temperature of 9. 1 M solutions Unknowns: Number the beakers. Results Table. Burn the loop end of the wire to remove any dust. Most people probably remember doing this experiment in school chemistry lessons, if not with the full range of ions shown here, but for the uninitiated a brief explanation of the origin of the colours follows. Calculate the energy of a photon. This lab is provided as a hands on way to test an unknown for the identity of cation and anion by using a flame; Question: Module 5 Lab Report - CHEM 1806 Anions, Cations, and Ionic Reactions Follow the directions in the lab write up. Bookmark File PDF Flame Test Lab 10 Answer Key Flame Test Lab Activity Key flame test. Use Table 1 in the Background section to record the approximate wavelength of light emitted for each metal in Data Table 2. Procedure: 1. Post-Lab Questions 1. Ask Expert Tutors You can ask You can ask You can ask (will expire ). Complete the: Flame test virtual lab; Answer the Pre and Post lab questions: Flame test with lab write-up; Complete a Lab write-up; Electron configuration notes. Pre-Lab Questions: 1. 89% average accuracy. This part of the lab is an extension from day 1: Colors of the Rainbow ( Flame test lab) where students will elaborate on their findings. Part I Procedure. This was done by putting several known solutions on different wooden splints and putting them over a fire to see. Most people probably remember doing this experiment in school chemistry lessons, if not with the full range of ions shown here, but for the uninitiated a brief. Carefully add (dropwise) 1% neutral. Electron Energy And Light Worksheet Answer KeyTitle. When heat is applied to the atoms, some electrons can have high energy levels and fall to the lower levels. Post Lab Calculations and Questions. This will be used to fill a test tube with blood. Record the wavelengths in meters in the. This test works well for metal ions, and was perfected by Robert Bunsen (1811 – 1899). Here are some of our favorite virtual lab activities for the classroom. Flame Test Lab Worksheet Answer Key with 22 Best Chemistry Unit 4 Review Images On Pinterest. Use dilute or approximately 0. 4 11/20/15 Pre-Lab Questions. The answers to the Sadlier Oxford Vocabulary Workshop Level B include the definitions for words like feud, haggle, hardy, legacy, and mirth. Do NOT add rows to the data tables. The color of the light depends on the frequency of the light given off. Turn the collar on the Bunsen burner so that you have an invisible or pale blue flame. Convert each of the wavelengths in the Data Table from nanometers to meters. Price: FREE. Flame test post lab answer key - sjyp. Informal together with feedback sessions help do away with minor splinters that may hamper the practice of achieving the vision. Learn vocabulary, terms, and more with flashcards, games, and other study tools. In order to complete How are elements identified by using a flame test? A metal salt is a compound of a metal and a nonmetal. Flame Test Lab (Honors Chemistry) BACKGROUND. This test works well for metal ions, and was perfected by Robert Bunsen (1811 – 1899). The goal of this lab is for you to discover some of the properties of ionic compounds. Test Answer Key In this site is not the thesame as a answer encyclopedia you' ' chemistry atomic structure test answer key taftaf de june 20th, 2018 - read and download chemistry atomic structure test answer key free ebooks in pdf format vocabulary for the college bound student answer key online jefferson math '. Flame Test Post Lab Answer Key. Metal Ion Color of Flame Calcium. What are alkaline earth metal elements? Where are they located on the periodic table? Procedure 1. Light the Bunsen burner. Label six clean, dry test tubes with the name of the substance to be tested. Flame Test Lab Worksheet Answer Key with 22 Best Chemistry Unit 4 Review Images On Pinterest. Lab Electron Configuration of Atoms and Ions Honors Chemistry from flame test lab worksheet answer key , source:yumpu. Lab 6, Experiment 2: Flame Tests: Using the color of a salt in a flame to identify certain metal ions. Purpose: To Observe The Characteristic Colors Produced By Metallic Ions When Heated In A Flame. Most people probably remember doing this experiment in school chemistry lessons, if not with the full range of ions shown here, but for the uninitiated a brief explanation of the origin of the colours follows. Basically, this means trying to find patterns of the flame color when. * This simulation take the spectral data from NATIONAL INSTITUTE OF STANDARDS AND TECHNOLOGY. see results table above. Take seven wooden splints that have been soaked. To do a flame test on a metallic element, the metal is first dissolved in a solution and the solution is then held in the hot, blue flame of a Bunsen burner. I need three sources of error, and so far I have this one: Even when cleaning the wire off thoroughly, small, residual amounts of liquid may still contaminate the wire, thus altering the shade of the color that is emitted by the heated substance. Carefully add (dropwise) 1% neutral. *If you were absent for either of the replacement reaction labs, download the directions and complete the lab by following the directions but looking up the different reactions on youtube to use as your observations. pdf - Flame Test Lab Questions Answer Key 1 Flame Test Lab. Played 111 times. February 6, 2014. Metal Color of Flame Data Table 2 Metal/Color of Flame λ (nm) λ (m) ΔE (J) Post-Lab Questions 1. Is the flame color a test for the metal or for the chloride in each compound? Explain your answer? 2. All plants need water, minerals, carbon dioxide, sunlight, and living space. Post author. 20 Flame Test Lab. it The Flame Test lab was an in-class lab where we tested chemicals in the flames to see the wide range of colors in the color spectrum. Flame Test Lab Worksheet Answer Key with 22 Best Chemistry Unit 4 Review Images On Pinterest. pdf FREE PDF DOWNLOAD The energy levels in atoms and ions are the key to the production and detection of light. color in the flame test, flame test lab questions answer key, flame test pre lab questions write out the electron, la09 laboratory report laboratory assignment 9 electron, chemistry flame test lab flashcards quizlet, flame tests atomic emission and electron energy levels, academic chemistry lab flame tests mrs tarpey s,. An air-rich or fuel-lean (hot) flame is much more desirable. Periodic Properties Lab with lab write-up; Light waves introduction lesson with notes. What key IR and NMR absorptions would allow you to determine that the product from the first reaction above has been successfully synthesized?. Complete the: Flame test virtual lab; Answer the Pre and Post lab questions: Flame test with lab write-up; Complete a Lab write-up; Electron configuration notes. Place the test tube containing the sugar in a test tube holder. While the light emitted from a few excited metal ions is beautiful, in the laboratory a simple flame test is often very helpful in identifying an unknown metal ion. The physical properties of a substance such as flame color, crystal structure, solubility, conductivity and melting point of a substance tell us a lot about the type of bonding in a compound. Use flame tests to identify a metal or metallic salt by the color that it produces when it is put into a flame. After calculating and then preparing specific molarity solutions of strontium chloride, copper II chloride and potassium chloride (good practice!), students observe the distinct colors. Flame Test Lab PURPOSE: Using an elements spectrum, learning how the electrons move around in the atom. Price: FREE. This test works well for metal ions, and was perfected by Robert Bunsen (1811 - 1899). Activation Energy Electron Configuration POGIL - Answers. Learn our geolocator map will find the ny notary acknowledges this online class at. Use dilute or approximately 0. Bookmark File PDF Flame Test Lab 10 Answer Key Flame Test Lab Activity Key flame test. And to learn why light is shown as the color it is by the excitement of electrons jumping from one shell to the next. 89% average accuracy. Answers are shuffled randomly, you will never get the same questions in this MO permit exam prep app. The purpose of this experiment was to observe the emission spectrum of atoms in a flame test. 7 Atomic Emission Spectrum: Atoms Emit Energy Electrons return to lower energy levels when 15 Flame Test The excited electron produces one or more specific lines in the visible light 23 Data Analysis and Conclusion Answer the data analysis and conclusion questions in your lab notebook. pdf Download Selected Download. General Chemistry. Explain that some wavelengths of light are in the visible light area, while others are not, but can still cause chemical reactions. Part I Procedure. Each word document, safety answer key is for the gaps using the gaps only, scientific method name science lab safety symbols. br chemfax chemical formulas kit lab answer key Pdf to read on the plane or the commuter train, whereas print books are heavy and chemfax lab chemical formula kit answers - Bing Flinn Scientific is the #1 source for science supplies and equipment both in and outside the classroom. This test works well for metal ions, and was perfected by Robert Bunsen (1811 – 1899). A technician will insert a small needle into a vein on your arm or hand. The further away from the nucleus they are, the more energy the electrons have. So I need two more, I considered the fact that a lot of times the. To test conductivity in this lab, we will dissolve our substances in water. The goal of this lab is for you to discover some of the properties of ionic compounds. The purpose of the flame test lab is to observe the characteristics colors produced by certain metallic Ions when vaporized in a flame. The trouble comes when fibers are blends. Hold the splint in a low bunsen burner flame and record the main color that is emitted (other than the color of the flame) in. Browse flame test electron configuration resources on Teachers Pay Teachers, a marketplace trusted by millions of teachers for original educational resources. All plants need water, minerals, carbon dioxide, sunlight, and living space. Also, learning how someone could use the spectrums of the elements to identify an unknown. In this lab you will learn how to light and adjust a burner flame and to locate the hottest part of the flame. com Art perfect flame gas grill from ash to flame women rising soul of flame imdalind 4 flame test lab answer key flame test lab worksheet flame wood stove manual autodesk flame user guide country flame wood stove country flame pellet stove flame of recca episode guide char broil flame tamer. Calculate the quantity of heat, in kilojoules, (a) required to raise the temperature of 9. The Flame Test lab was done in several parts. Flame Test Lab Pre-Lab Answers (18 pts)/ Answer in complete sentences Post Lab Answers (50 pts)/ Answer in complete sentences. In Classwork. pdf Download Selected Download. Virtual Lab Challenge: Separation of Mixtures (POST LAB) DRAFT. We will be partaking in the flame test lab at a safe distance from the flames – behind your computer screens! Please watch the video below. An unknown solution gives a brick-red flame test. The answers to the Sadlier Oxford Vocabulary Workshop Level B include the definitions for words like feud, haggle, hardy, legacy, and mirth. Virtual Lab Challenge: Separation of Mixtures (POST LAB) DRAFT. The further away from the nucleus they are, the more energy the electrons have. By Compound Interest. Would we expect ionic and/or covalent substances to conduct electricity when dissolved in water? Explain your answer. If the flame tester has to be assembled, it is recommended that two sheets are used, one for putting together the tester and the other sheet. Of different metals that we would Test and then use the speed of is Metals, to identify a couple of the flame Test Part 1: pre-lab Introduction: in the. Flame Test Lab PURPOSE: Using an elements spectrum, learning how the electrons move around in the atom. This set covers a unit on cell structure and physiology. Post author. Carolina Solution Sheets. pdf Download Selected Download. To do a flame test on a metallic element, the metal is first dissolved in a solution and the solution is then held in the hot, blue flame of a Bunsen burner. CAUTION: too much air can extinguish the flame. You could readily identify the elements that had obvious colors different form all the others- such as copper that gave off a blue/green color and lithium that gave off a bright red color. Dip the loop into the lithium salt solution. Periodic Properties Lab with lab write-up; Light waves introduction lesson with notes. Perform a flame test on an unknown metal chloride and record its characteristic color(s) and the probable identity of the unknown in the Data Table. After watching the video, please answer the questions below. Answer key for Light Properties WS Flame Test Lab. 5 flame tests and atomic spectra experiment. To do a flame test on a metallic element, the metal is first dissolved in a solution and the solution is then held in the hot, blue flame of a Bunsen burner. This test works well for metal ions, and was perfected by Robert Bunsen (1811 – 1899). In the matching section of the test the answers are E Do you have unit 2 and 3? Unit 1 Answers; About Me. • Perform the flame tests on the known solutions: Dip the wire into your first solution and place it in the hottest part of the Bunsen burner flame. When placed in a flame, aluminum does not change the flame's color, and so a visual flame test cannot be used to show the presence of Al. flame test pre lab answer key. Complete the: Flame test virtual lab; Answer the Pre and Post lab questions: Flame test with lab write-up; Complete a Lab write-up; Electron configuration notes. Your post might reference some of this work to support statements you make. 1) Does your reaction have oxygen as one of it's reactants • In a flame test, copper ions emit a deep blue-green light, much more blue than the flame test for barium. Data Table. The purpose of this lab was to observe the effect of certain compounds and more specifically elements in the compounds being burned. Flame Test Lab. You should perform the test a few times for each solution. A fuel-rich (cool) flame is yellow and sooty. In this post I described the materials I used in my first unit of the school year. Unformatted text preview: 11/6/12 Lab 2 PostLab -‐‑ Flame Tests WebAssign Lab 2 PostLab -­ Flame Tests (Postlab) Current Score : 23 / 25 Due : Thursday, September 6 2012 11:00 PM EDT The due date for this assignment is past. This was done by putting several known solutions on different wooden splints and putting them over a fire to see. LAB: PROPERTIES OF IONIC COMPOUNDS (50pts) Introduction. To test this idea, the scientist set up an experiment. Flame test post lab questions answers keyword after analyzing the system lists the list of keywords related and the list of websites with related content, in addition you can see which keywords most interested customers on the this website. Dilute ammonia is added to that precipitate but the precipitate remains. Bookmark File PDF Flame Test Lab 10 Answer Key Flame Test Lab Activity Key flame test. In Classwork. Learn vocabulary, terms, and more with flashcards, games, and other study tools. What are alkaline earth metal elements? Where are they located on the periodic table? Procedure 1. Learn how in 5 minutes with a tutorial resource. ) Light a wood splint and place inside the test tube. So I need two more, I considered the fact that a lot of times the. 25 kg of water from 22. Then, place one of the saturated sticks into the flame. The Nature of Flame Test Lab Worksheet Answer Key in Studying. pdf Download Selected Download. It was difficult to identify a couple of the elements that had colors that were similar. New research provides key insight about mitochondrial replacement therapy A discovery published in 2016 may unlock the answer to a vexing scientific question: How to conduct mitochondrial replacement therapy, a new gene-therapy technique, in such a way that safely prevents the transmission of harmful mitochondrial gene mutations from mothers to. touch the sides of the test tube or the liquid with the litmus paper. This test works well for metal ions, and was perfected by Robert Bunsen (1811 – 1899). Mystery Science. In this lab you will learn how to light and adjust a burner flame and to locate the hottest part of the flame. Flame Test Lab Questions Answer Key: 1. Pre Lab Flame Tests Lab in your lab notebook Finish Calculations for light properties worksheet for homework if you did not complete it in class. nova official website search for the super battery. Pre-laboratory Assignment: Flame Tests of Metal Cations. Results Table. Basically, this means trying to find patterns of the flame color when. com Art perfect flame gas grill from ash to flame women rising soul of flame imdalind 4 flame test lab answer key flame test lab worksheet flame wood stove manual autodesk flame user guide country flame wood stove country flame pellet stove flame of recca episode guide char broil flame tamer. Analysis Of Cations And Anions Group IV I. Place into each test tube 5 drops of the appropriate hydrocarbon. To Identify An Unknown Metallic Ion By Means Of Its Flame Test. Answers in as fast as 15 minutes. None of the Bg atoms in the original sample would have the same amount of mass as the calculated atomic mass of the element because because the atomic mass is the weighted average of all the Bg atoms. Label six clean, dry test tubes with the name of the substance to be tested. Flame Test Post Lab Answer Key. Materials and Equipment:. Read Paper. Ask Expert Tutors You can ask You can ask You can ask (will expire ). An unknown solution gives a brick-red flame test. This part of the lab is an extension from day 1: Colors of the Rainbow ( Flame test lab) where students will elaborate on their findings. To write your Answers in your text and answer questions in complete sentences question segment and are bold. Dip the loop into the lithium salt solution. This test works well for metal ions, and was perfected by Robert Bunsen (1811 – 1899). Complete the: Flame test virtual lab; Answer the Pre and Post lab questions: Flame test with lab write-up; Complete a Lab write-up; Electron configuration notes. senior chemistry extended experimental investigations. What are the alkali metal elements? Where are they located on the periodic table? 2. Hold the splint in the flame and record the color of the flame that is produced. To do a flame test on a metallic element, the metal is first dissolved in a solution and the solution is then held in the hot, blue flame of a Bunsen burner. A fuel-rich (cool) flame is yellow and sooty. Note the color of the flame and record your observations on your data sheet. pdf Download Selected Download. By Compound Interest. Dilute ammonia is added to that precipitate but the precipitate remains. nova official website search for the super battery. Part One: Flame Tests (As an option, this could be a demo rather than a student activity) When solutions of metals are heated in a Bunsen burner flame, they give off characteristic colours. Be sure to show all work, round answers, and include units on all answers. Virtual Lab Challenge: Separation of Mixtures (POST LAB) DRAFT. Flame Test Post Lab Answer Key. Hold the splint in the flame and record the color of the flame that is produced. Question #258458 from lei. Your post might reference some of this work to support statements you make. The questions should be answered on a separate (new) page of your lab notebook. Place the test tube containing the sugar in a test tube holder. End of The Experiment : October 18th, 2014 at 10. Lab 6, Experiment 2: Flame Tests: Using the color of a salt in a flame to identify certain metal ions. [VIEW] Flame Test 1. What are alkaline earth metal elements? Where are they located on the periodic table? Procedure 1. 85kg aluminum bar (specific heat of aluminum = 0. The goal of this lab is for you to discover some of the properties of ionic compounds. As well as to identify unknown metallic ions by means of its flame test. When heat is applied to the atoms, some electrons can have high energy levels and fall to the lower levels. Start studying Flame Test Pre-Lab Quiz. Also, learning how someone could use the spectrums of the elements to identify an unknown. br chemfax chemical formulas kit lab answer key Pdf to read on the plane or the commuter train, whereas print books are heavy and chemfax lab chemical formula kit answers - Bing Flinn Scientific is the #1 source for science supplies and equipment both in and outside the classroom. Bookmark File PDF Flame Test Lab 10 Answer Key Flame Test Lab Activity Key flame test. Part I Procedure. Flame Test Lab Activity Key Note: If chloride compounds are not available, metal nitrate compounds may be substituted. Post Lab Calculations and Questions. Is the flame color a test for the metal or for the chloride in each compound? Explain your answer? 2. Purpose: To Observe The Characteristic Colors Produced By Metallic Ions When Heated In A Flame. Although the data entered in the sheets are to help one out, it can be a puzzle for one’s mind as well. Post-lab questions: Why is it important to test the flame color of the methanol without any compounds dissolved in it? Do the positive ions or the negative ions cause the change in flame color? Explain why based on your observations. LAB: PROPERTIES OF IONIC COMPOUNDS (50pts) Introduction. 2 Move any flammable materials away from work area 3 Attach the connecting hose. Carefully add (dropwise) 1% neutral. What is released when an electron loses energy? 3. This test works well for metal ions, and was perfected by Robert Bunsen (1811 – 1899). Bookmark File PDF Flame Test Lab 10 Answer Key Flame Test Lab Activity Key flame test. Of different metals that we would Test and then use the speed of is Metals, to identify a couple of the flame Test Part 1: pre-lab Introduction: in the. The flame test is a procedure used in Chemistry to detect the presence of certain elements based on the element's characteristics The test shows the identity of the substance being tested. PROCEDURE: Using a scoopula, place a small amount of the metal salt solution into a clean petri dish. Dilute ammonia is added to that precipitate but the precipitate remains. The purpose of this experiment was to observe the emission spectrum of atoms in a flame test. Get the app ». Chemists began studying colored flames in the 18th century and soon used "flame tests" to distinguish between some elements. Hold the splint in a low bunsen burner flame and record the main color that is emitted (other than the color of the flame) in. Although the data entered in the sheets are to help one out, it can be a puzzle for one's mind as well. docx Answer Key. FLAME TEST LAB. Bookmark File PDF Flame Test Lab 10 Answer Key Flame Test Lab Activity Key flame test. 89% average accuracy. 6 Comments. Metal/Flame Color λ (nm) λ (m) ΔE (J) Post-Lab Questions. Get eight dishes and label them as Stannous Chloride, Lead Nitrate, Cupric Chloride, Calcium Chloride, Potassium Chloride, Magnesium Chloride, Sodium Chloride, and Barium Chloride. Turn the collar on the Bunsen burner so that you have an invisible or pale blue flame. A scientist wanted to test the effectiveness of different fertilizers in supplying needed minerals to plants. Most salts contain a metal and a non-metal. Adhere about what to edit to the directions. Basically, this means trying to find patterns of the flame color when different elements are burned. Experiment Title : Analysis of Cations and Anions II. color in the flame test, flame test lab questions answer key, flame test pre lab questions write out the electron, la09 laboratory report laboratory assignment 9 electron, chemistry flame test lab flashcards quizlet, flame tests atomic emission and electron energy levels, academic chemistry lab flame tests mrs tarpey s,. Background. Safety: Wear goggles and an apron. New research provides key insight about mitochondrial replacement therapy A discovery published in 2016 may unlock the answer to a vexing scientific question: How to conduct mitochondrial replacement therapy, a new gene-therapy technique, in such a way that safely prevents the transmission of harmful mitochondrial gene mutations from mothers to. What are the alkali metal elements? Where are they located on the periodic table? 2. Analysis Of Cations And Anions Group IV I. Most people probably remember doing this experiment in school chemistry lessons, if not with the full range of ions shown here, but for the uninitiated a brief explanation of the origin of the colours follows. End of The Experiment : October 18th, 2014 at 10. Convert each of the wavelengths in the Data Table from nanometers to meters. This test works well for metal ions, and was perfected by Robert Bunsen (1811 – 1899). Flame Test Lab Worksheet Answer Key Did You Hear About Math Worksheet Answer Key are a type of education aid. Flame test with lab write-up; To make-up Flame Test Lab. The following are links to examples of well researched lab report papers that will help you to know more on how to write a conclusion for a lab report. Basically, this means trying to find patterns of the flame color when different elements are burned. Aseptic Transfer Of Bacteria: FOR THIS LAB YOU WILL BE WORKING INDIVIDUALLY. Though the actual flame test cannot be done, through this virtual lab you will observe the flame through the colors in the following photos. Record any change in the color of the solution and the litmus paper. This graphic looks at the colour of various metal and metalloid ions that occur during flame tests. Learn our geolocator map will find the ny notary acknowledges this online class at. Different elements burn with different colored flames. For example, sodium makes the flame turn bright yellow - this is the same yellow colour made by sodium street lamps and many fireworks. Grasp one presoaked toothpick with forceps. Materials and Equipment:. To test conductivity in this lab, we will dissolve our substances in water. To do a flame test on a metallic element, the metal is first dissolved in a solution and the solution is then held in the hot, blue flame of a Bunsen burner. *If you were absent for either of the replacement reaction labs, download the directions and complete the lab by following the directions but looking up the different reactions on youtube to use as your observations. Bookmark File PDF Flame Test Lab 10 Answer Key Flame Test Lab Activity Key flame test. You will be provided with Staphylococus epidermidis in a broth and on a plate. Burn the loop end of the wire to remove any dust. Instrumental methods of analysis are faster, and more accurate and more sensitive than simple chemical tests. Procedure: 1. Flame Test Lab Pre-Lab Answers (18 pts)/ Answer in complete sentences Post Lab Answers (50 pts)/ Answer in complete sentences. February 6, 2014. pdf - Flame Test Lab Questions Answer Key 1 Flame Test Lab. Flame Test Lab Pre-AP Mr. The secondary purpose of the lab was to identify unknown compounds that we would test and then guess as to what they were. Experiment Title : Analysis of Cations and Anions II. Ask Expert Tutors You can ask You can ask You can ask (will expire ). Some electrons emit as much energy as the energy level difference. Convert each of the wavelengths in the Data Table from nanometers to meters. The trouble comes when fibers are blends. Take seven wooden splints that have been soaked. Flame test with lab write-up; To make-up Flame Test Lab. In addition to this, a printable 22 page activity booklet contains student activities. To write your Answers in your text and answer questions in complete sentences question segment and are bold. Answer Key. 3 Electron Orbitals and electron configurations Electron. Energy levels or "shells"exist for electrons in atoms Emission spectrum - Wikipedia, the free encyclopedia Lab: Flame Tests - Chemistry www. Also, learning how someone could use the spectrums of the elements to identify an unknown. Then answer the questions. In A Flame Test, The Element Will Give Off A Characteristic Color That Serves As A Simple Method Of Identification Of That Element. Most people probably remember doing this experiment in school chemistry lessons, if not with the full range of ions shown here, but for the uninitiated a brief. Then, in the 1830s, the Italians discovered that adding metal salts to the fireworks mixture resulted in interesting colors, just like in the flame test in this science activity. Part I Procedure. Atwood Hydro Flame Furnace - Scholarships. Why do different metals have different characteristic flame test colors? 3. We have tutors online 24/7 who can help you get unstuck. This graphic looks at the colour of various metal and metalloid ions that occur during flame tests. The following are links to examples of well researched lab report papers that will help you to know more on how to write a conclusion for a lab report. When heat is applied to the atoms, some electrons can have high energy levels and fall to the lower levels. conventional weapons atomic rockets projectrho com. Identify any odor that is readily apparent by wafting the fumes toward your nose. This set covers a unit on cell structure and physiology. Stay connected to all things IU. The trouble comes when fibers are blends. Answer Key. see results table above. Mystery Science. Use Table 1 in the. Carolina Solution Sheets. Your work can be viewed below, but no changes can be made. Unformatted text preview: 11/6/12 Lab 2 PostLab -‐‑ Flame Tests WebAssign Lab 2 PostLab -­ Flame Tests (Postlab) Current Score : 23 / 25 Due : Thursday, September 6 2012 11:00 PM EDT The due date for this assignment is past. This ended up being a set of 59 warm ups or pages that cover cell structure and function, photosynthesis, respiration, and.

Источник: http://e-commercemitgrips.de/flame-test-post-lab-answer-key.html

Open Access

Peer-reviewed

  • Zhe Zhao,
  • Riku Fagerlund,
  • Helena Tossavainen,
  • Kristina Hopfensperger,
  • Rishikesh Lotke,
  • Smitha Srinivasachar Badarinarayan,
  • Frank Kirchhoff,
  • Perttu Permi,
  • Kei Sato,
  • Daniel Sauter,
  • Kalle Saksela
  • Zhe Zhao, 
  • Riku Fagerlund, 
  • Helena Tossavainen, 
  • Kristina Hopfensperger, 
  • Rishikesh Lotke, 
  • Smitha Srinivasachar Badarinarayan, 
  • Frank Kirchhoff, 
  • Perttu Permi, 
  • Kei Sato, 
  • Daniel Sauter
PLOS

x

?

This is an uncorrected proof.

Abstract

The accessory protein Nef of human and simian immunodeficiency viruses (HIV and SIV) is an important pathogenicity factor known to interact with cellular protein kinases and other signaling proteins. A canonical SH3 domain binding motif in Nef is required for most of these interactions. For example, HIV-1 Nef activates the tyrosine kinase Hck by tightly binding to its SH3 domain. An archetypal contact between a negatively charged SH3 residue and a highly conserved arginine in Nef (Arg77) plays a key role here. Combining structural analyses with functional assays, we here show that Nef proteins have also developed a distinct structural strategy—termed the "R-clamp”—that favors the formation of this salt bridge via buttressing Arg77. Comparison of evolutionarily diverse Nef proteins revealed that several distinct R-clamps have evolved that are functionally equivalent but differ in the side chain compositions of Nef residues 83 and 120. Whereas a similar R-clamp design is shared by Nef proteins of HIV-1 groups M, O, and P, as well as SIVgor, the Nef proteins of SIV from the Eastern chimpanzee subspecies (SIVcpzP.t.s.) exclusively utilize another type of R-clamp. By contrast, SIV of Central chimpanzees (SIVcpzP.t.t.) and HIV-1 group N strains show more heterogenous R-clamp design principles, including a non-functional evolutionary intermediate of the aforementioned two classes. These data add to our understanding of the structural basis of SH3 binding and kinase deregulation by Nef, and provide an interesting example of primate lentiviral protein evolution.

Author summary

Viral replication depends on interactions with a plethora of host cell proteins. Cellular protein interactions are typically mediated by specialized binding modules, such as the SH3 domain. To gain access to host cell regulation viruses have evolved to contain SH3 domain binding sites in their proteins, a notable example of which is the HIV-1 Nef protein. Here we show that during the primate lentivirus evolution the structural strategy that underlies the avid binding of Nef to cellular SH3 domains, which we have dubbed the R-clamp, has been generated via alternative but functionally interchangeable molecular designs. These patterns of SH3 recognition depend on the amino acid combinations at the positions corresponding to residues 83 and 120 in the consensus HIV-1 Nef sequence, and are distinctly different in Nef proteins from SIVs of Eastern and Central chimpanzees, gorillas, and the four groups of HIV-1 that have independently originated from the latter two. These results highlight the evolutionary plasticity of viral proteins, and have implications on therapeutic development aiming to interfere with SH3 binding of Nef.

Citation: Zhao Z, Fagerlund R, Tossavainen H, Hopfensperger K, Lotke R, Srinivasachar Badarinarayan S, et al. (2021) Evolutionary plasticity of SH3 domain binding by Nef proteins of the HIV-1/SIVcpz lentiviral lineage. PLoS Pathog 17(11): e1009728. https://doi.org/10.1371/journal.ppat.1009728

Editor: Raul Andino, University of California San Francisco, UNITED STATES

Received: June 17, 2021; Accepted: October 28, 2021; Published: November 15, 2021

Copyright: © 2021 Zhao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: KS was funded by the Helsinki University Central Hospital Research Council grant TYH2017248 and by the Jane and Aatos Erkko Foundation (JAES2016). PP was supported by grants from the Academy of Finland (323435) and Jane and Aatos Erkko Foundation (JAES2019). DS was supported by the Heisenberg Program (SA 2676/3-1) and the Priority Program SPP1923 (SA 2676/1-2) of the German Research Foundation (DFG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Primate lentiviruses comprise human immunodeficiency viruses (HIV-1 and HIV-2), as well as more than 40 simian immunodeficiency viruses (SIVs) infecting non-human primate species from sub-Saharan Africa. HIV-1 groups M, N, O, and P originate from four independent transmissions of SIV from Central chimpanzees (SIVcpzP.t.t.) and gorillas (SIVgor) to humans, whereas HIV-2 groups A to I are derived from nine zoonotic transmissions of SIV from sooty mangabeys (SIVsmm) (reviewed in [1]). SIVsmm was also transmitted to macaques, giving rise to SIVmac. The global AIDS pandemic is largely caused by HIV-1 group M strains, which are further divided into 10 subtypes (A, B, C, D, F, G, H, J K, and L), as well as several sub-subtypes and circulating recombinant forms [2–4].

The viral protein Nef is a multifunctional accessory factor encoded by all primate lentiviruses. While nef-defective HIV and SIV are replication-competent, infections with such viruses are associated with low viral loads and no or delayed pathogenesis in humans or experimentally infected macaques [5]. Nef itself exhibits no enzymatic activity but instead modulates host cell function by interacting with a plethora of other proteins to hijack cellular signaling and membrane trafficking pathways [6–8].

A function of HIV-1 Nef that is well understood at the molecular and mechanistic level is its interaction with Hck, a member of the Src-family tyrosine kinases. Nef tightly binds to the SH3 domain of Hck, thereby shifting it from an intramolecular autoinhibitory state into a catalytically active conformation [9,10]. Hck plays an important role in regulating activation and effector functions of macrophages, the second main target cell population of HIV-1 besides CD4+ T cells [11,12]. Many Nef functions in other cell types including T lymphocytes, (e.g., MHC-I downregulation [13], interfering with Lck localization and immunological synapse formation [13], and reorganization of the actin cytoskeleton [14,15]) are also strictly dependent on its SH3 binding capacity. However, the relevant SH3 proteins in these cells do not bind to Nef as tightly as Hck, and have therefore remained less well characterized [7].

The discovery of the Nef-Hck interaction provided the first example of pathogen takeover of host cell signaling via SH3 domain ligand mimicry [16]. Src Homology 3 (SH3) domains are short (~60 aa) modular protein units specialized for mediating protein interactions via proline-rich core binding sites in the target proteins, and are ubiquitous (~300 in human) in eukaryotic proteins involved in regulation of cell behavior [17,18].

Studies on the Nef-SH3 complex also revealed a new paradigm for the structural basis of SH3 binding: It could be shown that docking of the proline-rich (PxxP) peptide of Nef by the SH3 core binding interface is assisted by further molecular contacts of the so-called SH3 RT-loop region to provide additional specificity and affinity to this interaction [9]. The amino acid residues that form a binding pocket for the RT-loop of the Hck-SH3 domain are highly conserved in HIV-1 and HIV-1-like Nef proteins, and provide them with a capacity for strong binding to Hck [9,19]. Nef proteins from the HIV-2/SIVsmm groups lack these residues and hence Hck binding capacity, but as evidenced by the SIVmac293-Y113W/E117T/E118Q triple mutant [20], can acquire this function upon introduction of just a few key residues from HIV-1 Nef.

We have recently shown that Hck activation by Nef leads to Raf/MAPK pathway activation and triggers the secretion of proinflammatory cytokines [19,21]. In agreement with their enhanced affinity for Hck [9,20], we found that only HIV-1/SIVcpz type Nef proteins but not HIV-2/SIVsmm type Nef proteins show this function. The current study was incited by our observation that an HIV-1 group N Nef (clone YBF30) was unable to bind and activate Hck and the Raf/MAPK pathway for reasons that could not be readily explained by its amino acid sequence. Our investigation into this issue led to the discovery of a structural arrangement that we have termed the arginine (R)-clamp and characterized here. We show that different but functionally equivalent R-clamps have emerged in the HIV-1/SIVcpz lineage to coordinate Hck SH3 domain binding by Nef. This finding further highlights the importance of Nef-mediated SH3 binding and the enormous plasticity of primate lentiviral accessory proteins.

Results

In our previous studies we examined the capacity of Nef to activate Hck by monitoring tyrosine phosphorylation of paxillin, a prominent substrate for Src family tyrosine kinases, and by measuring the induction of AP-1-regulated reporter gene expression following Hck-activated MAPK signaling [19,21]. When additional Nef proteins from HIV-1 groups N, O, and P were examined (Fig 1), we unexpectedly noticed that the Nef protein of HIV-1 N YBF30 (AJ006022) was unable to induce paxillin phosphorylation or AP-1 activity despite sharing the 72PxxPxR77 motif (numbering based on the HIV-1 Nef consensus) known to be important for Hck binding by HIV-1/SIVcpz type Nefs (Fig 2). Importantly, YBF30 Nef was clearly expressed in these cells (Fig 1A), and as also shown previously [22] was functional when tested for its capacity to downregulate cell surface expression of CD4 (Figs 1C and S1).

thumbnail
Download:

Fig 1. Hck-activating potential of selected HIV-1 Nefs.

(A) Hck-expressing HZ-1 cells were co-transfected with vectors for paxillin and Nef from the indicated HIV-1 group M, N, O or P clones, or mutants thereof. Lysates of the transfected cells were analyzed by Western blotting using antibodies against paxillin (PXN), phosphorylated paxillin (pPXN) and Nef. (B) HZ-1 cells were transfected with an AP-1 transcription factor-driven luciferase reporter alone (Control) or together with the indicated Nefs variants. Luciferase activity was measured in cells harvested 24 h post-transfection, and normalized to the corresponding control sample that was set to 1. The data shown are derived from three independent experiments, with SE indicated by error bars. The amino acid at position 120 of each Nef protein is indicated, aromatic residues are shown as a black 1-letter symbol in a white sphere, and isoleucine as a white letter in a black sphere. (C) Jurkat T cells stably expressing CD4 were infected with lentiviral vectors expressing GFP alone (No Nef) or together with plasmids expressing the indicated Nefs. CD4 down-regulation by Nef was measured using flow cytometry 48 h after lentiviral transduction, and histograms illustrating cell surface levels of CD4 among the GFP positive Jurkat cells are shown. Original dot plots are shown in S1 Fig.

https://doi.org/10.1371/journal.ppat.1009728.g001

thumbnail
Download:

Fig 2.

HIV-1 M consensus Nef amino acid sequence (A) and an alignment of the conserved central region (underlined in A) of selected Nef proteins from different primate lentiviruses (B). The key residues of the core SH3 docking site (PxxPxR motif), including R77, are highlighted in light blue. Residues involved in a binding pocket for the Hck SH3 domain RT-loop that are conserved in HIV-1 and HIV-1-like Nef proteins are highlighted in yellow. Residues at positions 83 and 120 forming the R-clamp coordinating the positioning of R77 are shown in bold and colored according to their side chain properties (small in green, hydrophilic in blue, aliphatic or methionine in red, aromatic in brown). The names of HIV/SIV strains from which Nef proteins were included in this study are shown on the left.

https://doi.org/10.1371/journal.ppat.1009728.g002

To examine if this is a characteristic of several HIV-1 group N viruses we tested three additional HIV-1 N Nef proteins from the strains 02CM-DJO0131 (AY532635), YBF106 (AJ271370), and S4858 (KY498771). However, we found that all of them were fully competent for Hck activation, and induced paxillin phosphorylation and AP-1 activity as efficiently as Nef from the widely used HIV-1 group M strain SF2 (Fig 1).

Since the core SH3 docking motif (72PxxPxR77) as well as the key RT-loop accommodating residues, including F90, W113, T117, and Q118, were conserved in YBF30 Nef (see Fig 2) we had a more detailed look at the original X-ray structure of the HIV-1 Nef-SH3 complex (1EFN; [9]), which directed our attention to Nef residue 120. This position is occupied by an aromatic residue (tyrosine or phenylalanine) in virtually all HIV-1 M Nef proteins, while an isoleucine residue is found in this position in YBF30 Nef (Fig 2). In the 1EFN structure, Y120 can be seen to coordinate the Nef-SH3 interaction by buttressing the side-chain of the R77 in the Nef 72PxxPxR77 motif together with the SH3 residue W37 (SH3 numbering according to [18]) in order to stabilize the close positioning of R77 with the acidic SH3 residue D17 to form a critical salt bridge with it (Fig 3A and 3B). We termed this steric guiding of the Nef residue R77 into close proximity of D17 in Hck-SH3 the “arginine (R)-clamp”, and hypothesized that the failure of YBF30 Nef to activate Hck is due to the failure of its Ile120 residue to from a functional R-clamp (Fig 3C).

thumbnail
Download:

Fig 3. The arginine clamp.

(A) Structure of the HIV-1 Nef (green)–SH3 (pink) complex (PDB ID 1EFN) highlighting critical residues in the interaction interface. (B) Close up view of the R clamp, stabilized by stacked Y-R-W side chains and a salt bridge between R77 (Nef) and D17 (SH3) side chains. (C) In the Y120I structure, one side of the R77 guanidinium plane is lacking non-bonded interactions. (D) In the A83M/Y120I, structure the long methionine side chain re-establishes non-bonded contacts for R77. The presented mutated complex structures were created by replacing Y120 or Y120 and A83 in UCSF Chimera [45] and minimizing the structures with AMBER [46].

https://doi.org/10.1371/journal.ppat.1009728.g003

In support of this hypothesis, we found that YBF30 Nef became fully competent for Hck activation when its I120 residue was replaced by a tyrosine (YBF30 I120Y) (Fig 1). We then further investigated this idea by changing the Y120 residue in the Nef protein from the HIV-1 M laboratory strain SF2 into an YBF30-like isoleucine (Nef mutant SF2 Y120I) or into a phenylalanine, the other commonly found residue at this position (Nef mutant SF2 Y120F). While the conservative Y120F amino acid change did not affect Hck activation, the Y120I mutation recapitulated the failure of YBF30 Nef to stimulate paxillin phosphorylation and AP-1 activity, without substantially compromising protein expression levels or CD4 downregulation (Fig 1). Thus, we conclude that the lack of a functional R-clamp due to the I120 residue likely explains the inability of YBF30 Nef to activate Hck.

Analysis of additional HIV-1 group N Nefs revealed that in 5 out of the total of 12 sequences that are available in databases the position 120 was occupied by Ile (Table 1). This is in stark contrast to the Nef proteins from the three other HIV-1 groups M, O, and P, in which residue 120 is invariably Y or F. We had access to another I120-containing HIV-1 N Nef namely 2693BA (GQ925928), and went on to examine its ability to activate Hck. Having already established a strict correlation between paxillin phosphorylation and AP-1 activity as markers of Hck activation (Fig 1 and [19]), we chose to focus on using AP-1-driven reporter gene expression as the read-out in our further studies. Unexpectedly, we found that despite carrying the I120 residue 2693BA Nef had an undiminished capacity to activate Hck (Fig 4). In order to understand this finding, we returned to the Nef-SH3 structure 1EFN, and noted the involvement of Nef residue 83 in contacting the critical R-clamp residue 120 (Fig 3). Residue 83 is an amino acid with a tiny (Ala or Gly) or a hydrophilic side-chain (Gln, Ser, Asp, or Glu) in virtually all Nef proteins that have an aromatic (Phe or Tyr) residue at position 120. However, in 2693BA Nef position 83 is occupied by methionine, a residue with a large hydrophobic/aliphatic side-chain. We therefore hypothesized that M83 might compensate for the presence of isoleucine instead of a planar aromatic residue at position 120 (Figs 3D and S4), and that the M83/I120 residue pair of 2693BA Nef might be functionally equivalent to the A83/F120 pair found in the Hck-activating HIV-1 N Nef proteins 02CM-DJO0131 and YBF106.

thumbnail
Download:

Fig 4. Role of residue 83/120 pairing in the HIV-1 N Nef R-clamp.

(A) HZ-1 cells were transfected with an AP-1-dependent luciferase reporter alone (Control) or together with wild type (WT) versions of YBF30 or 2693BA Nef, or mutants thereof carrying reciprocal amino acid changes of residue 83 (YBF30 Q83M and 2693BA M83Q). Luciferase activity was measured from cells 4 h post-transfection, and normalized to the corresponding control sample that was set to one. The data shown are derived from three independent experiments, with SE indicated by error bars. The amino acid combinations at positions 83 and 120 are indicated here and in the figures below as single-letter symbols and color-coded according to the residue classification shown in Table 1. Specifically, small (G or A) or hydrophilic residues (D, E, Q, or S) at position 83 are shown as a black 1-letter symbol in a white sphere (here Q83 in YBF30), whereas aliphatic or methionine residues at this position are shown as a white letter in a black sphere (here M83 in 2693BA). At position 120, aromatic residues (F or Y) are shown as a black letter in a white sphere (not found in YBF30 or 2693BA), whereas isoleucine is shown as a white letter in a black sphere (here I120 in YBF30 and 2693BA). (B) CD4 downregulation by Nef in stably transduced Jurkat T cells was examined as in Fig 1C. Original dot plots are shown in S2 Fig.

https://doi.org/10.1371/journal.ppat.1009728.g004

To test this idea, we swapped residues 83 between the I120-containing 2693BA and YBF30 Nef proteins. In line with our hypothesis, this reversed the differences in the Hck-activating capacity of these HIV-1 N Nefs, such that Nef-2693BA lost its ability to stimulate AP-1 activity, whereas YBF30 Nef became fully competent for this function (Fig 4). Thus, we conclude that Nef residues 83 and 120 appear to function together to form the R-clamp that coordinates Hck-SH3 binding by Nef, and that different combinations of residue pair 83/120 have evolved for building a functional R-clamp in HIV-1 group N Nefs.

Whereas among the Nef proteins of HIV-1, Ile can be found at position 120 only in group N viruses, we noted that several SIVcpz Nefs also contain Ile120 (Table 1). To test if our conclusions regarding the alternative R-clamp designs hold true also for SIVcpz Nef proteins, we examined five of these, three from SIV of the chimpanzee subspecies Pan troglodytes troglodytes (P.t.t.), namely MB897 (EF535994), CAM5 (AJ271369), and EK505 (DQ373065), and two from Pan troglodytes schweinfurthii (P.t.s.), namely Tan2 (EF394357) and Nok5 (AY536915).

We found that similar to the HIV-1 N YBF30 Nef, some of these SIVcpz Nefs (Cam5 and EK505) failed to activate Hck, whereas others (MB897, Nok5, and Tan2) were functional in this regard although all were able to downregulate CD4 (Fig 5). When the Hck activating capacity was compared with the residue 83/120 composition of these SIVcpz alleles, a perfect agreement was found with the structural R-clamp principles deduced from the results obtained with HIV-1 N Nefs. All SIVcpz Nef proteins that were competent for Hck activation contained a “permissible” [83] + [120] residue combination defined as [tiny/hydrophilic] + [Phe/Tyr] or [aliphatic] + [Ile], whereas both of the two non-activating SIVcpz Nef proteins contained a mixed pattern of amino acid classes at these positions (Figs 2 and 5).

thumbnail
Download:

Fig 5. R-clamp architecture of SIVcpz Nef proteins.

(A) HZ-1 cells were transfected with an AP-1-luciferase reporter alone (Control) or together with wild-type or the indicated mutants of Nef from SIV strains from Pan troglodytes troglodytes (MB897, Cam5, and EK505) or Pan troglodytes schweinfurthii (Nok5 and Tan2). Luciferase activity in the transfected lysates was analyzed as in Fig 1C. The data shown are derived from three independent experiments, with SE indicated by error bars. The amino acid combinations at positions 83 and 120 are indicated as single-letter symbols and color-coded as explained in Fig 4. (B) CD4 downregulation by Nef in stably transduced Jurkat T cells was examined as in Fig 1C. Original dot plots are shown in S3 Fig.

https://doi.org/10.1371/journal.ppat.1009728.g005

To further prove this concept, we showed that the failure of Cam5 and EK505 Nef to activate Hck could indeed be corrected by introducing a tyrosine residue at position 120 in these proteins, thereby providing them with an R-clamp residue combination 83 [hydrophilic] + 120 [Phe/Tyr]. Moreover, an E83M mutation (resulting in an 83 [aliphatic] + 120 [Ile] residue combination) conferred this activity to EK505 Nef. On the other hand, introducing a hydrophilic residue at position 83 of Nok5 Nef while maintaining its I120 residue (Nok5 I83Q) led to a complete loss of its Hck-activating potential, as predicted by our model on the compatible and non-compatible R-clamp residue combinations.

When the residue 83/120 composition was analyzed in a comprehensive survey of HIV-1/SIVcpz/SIVgor Nef sequences, it was interesting to observe that the different evolutionary lineages of primate lentivirus are associated with distinct R-clamp design strategies. In Table 1, these strategies have been grouped into four categories (I–IV) based on the amino acid side chain properties explained above, and their occurrence has been calculated as a percentage of all Nef sequences found for each lentivirus lineage. Whereas HIV-1 M, HIV-1 O, HIV-1 P, and SIVgor viruses fall almost exclusively into category I, HIV-1 N and SIVcpz viruses show more heterogeneity. Interestingly, however, all SIVcpz Nef proteins from the chimpanzee subspecies P.t.s. fall into category IV, while all SIVcpz(P.t.t.) Nef proteins are found in categories I (67%) or III (33%). HIV-1 N Nef proteins share the R-clamp design patterns with SIVcpz(P.t.t.) and SIVcpz(P.t.s.), and can be found spread between categories I, III, as well as IV. Finally, exclusively HIV-1 M Nef proteins can be found in category II, which includes a small but significant proportion (2.6%) of all the 5221 HIV-1 M Nef protein sequences in our survey.

Of note, the relative distribution of HIV-1 M Nef sequences into categories I to IV is not even between different viral subtypes. Whereas subtype B that constitutes more than half of all available HIV-1 M Nef sequences rarely falls in I120-containing categories III (0.31%) or IV (0.03%), this is much more common among subtype D and G Nef proteins, of which 2.91% and 2.59%, respectively belong to categories III or IV. And although only four of out the total 5221 HIV-1 M Nef sequences belong to category IV, two of these can be found among the 206 subtype D Nef sequences. On the other hand, while category II covers 2.6% of total HIV-1 M Nef sequences, this is the case for as many as 15.6% of subtype G and 7.1% of subtype H, but only 0.5% of subtype A strains.

Based on the experiments presented above it was inferred that Nef proteins in categories I, II, and IV can bind Hck to stimulate its tyrosine kinase activity, whereas category III Nef proteins with a “mixed” R-clamp residues pattern fail to do so. To further substantiate the generality of this conclusion, we introduced several additional individual and combinatorial 83/120 residue changes into the model Nef protein of HIV-1 M SF2 according to the classification presented in Table 1. We found that a Gln, Glu, or Ser residue could indeed all be introduced at position 83 of SF2-Nef without losing its Hck-activating function, as long as an aromatic residue was maintained at position 120 (Fig 6; SF2 A83Q, A83E, and A83S). Thus, all these different category II configurations could support a functional R-clamp when artificially introduced into SF2-Nef.

thumbnail
Download:

Fig 6. Comprehensive testing of R-clamp residue pairing rules by mutagenesis of SF2-Nef.

Nef-induced AP-1 activity in cells transfected without Nef (Control) or with the indicated wild-type or mutant versions of SF2 Nef was analyzed as in Fig 1B. The amino acid combinations at positions 83 and 120 of these SF2 Nef variants are indicated as single-letter symbols that are color-coded as explained in Fig 4.

https://doi.org/10.1371/journal.ppat.1009728.g006

By contrast, and again in full agreement with our R-clamp model, combination of these mutations with an Y120I change to introduce a category III “mixed” residue 83/120 pattern into SF2 (Q83S/Y120I, A83S/Y120I, and A83E/Y120I) resulted in all three cases in the loss of Hck activation. Finally, the disrupted Hck-activating function of the single residue 120 mutant SF2 Y120I could be fully rescued via an additional introduction of Met, Ile, or Leu at position 83 to generate artificial category IV-like SF2 Nef double mutants A83M/Y120I (“2693BA-like”), A83I/Y120I (“Nok5-like”), and A83L/Y120I (“Tan2-like”).

To confirm that the effects of the Nef R-clamp mutations on the observed cellular changes downstream of Hck activation were indeed due to altered formation of physical Nef-Hck complexes, we examined how wild-type and R-clamp mutated Nef proteins coprecipitated with Hck-p59 from transfected cells. As shown in Fig 7A, association with Hck was observed only for Nef proteins having a functional R-clamp, whereas category III R-clamp Nef proteins were as defective in this regard as the Nef-AxxA mutant carrying a fully disrupted SH3-binding motif.

thumbnail
Download:

Fig 7. Role of R-clamp residues 83/120 in intracellular Hck-Nef complex formation.

HEK293 cells were transfected with Myc-tagged wild-type HIV-1 M SF2 Nef (WT) or its mutants including Y120I, A83L-Y120I, P76A-P78A (AxxA), A83Q, and A83Q-Y120I) together with biotin acceptor domain-tagged wild-type Hck-p59 (WT) (A.) or its SH3 mutant Hck-C3 (B.). The Nef-contacting RT-loop residues centered around the critical SH3 amino acid 13 (Ile in WT and Asp in C3) are shown on top of these figures. Lysates of transfected cells were subjected to anti-Myc immunoprecipitation followed by Western blotting analysis of the immune complexes using labeled streptavidin (top panels). Equal Hck and Nef expression in the total lysates was confirmed by labeled streptavidin (Hck) (middle panels) or an anti-Myc (Nef) antibody (bottom panels).

https://doi.org/10.1371/journal.ppat.1009728.g007

In addition to its Class II binding motif (PxxPxR) a hydrophobic pocket in the core domain Nef is important for the Hck interaction by making contacts with the RT-loop region of Hck SH3 [9], in particular the isoleucine residue 13 (universal SH3 numbering according to [18]). Because the R-clamp residues 83 and 120 are part of the compact core of Nef, we wanted to exclude the possibility that mutations of these residues might influence Hck binding by disrupting the stabilizing contacts between Nef and I13 of Hck SH3. To this end, we constructed modified versions of Hck-p59 having artificial SH3 domains containing RT-loops that lack I13 and interact with Nef using diverse non-Hck-like molecular strategies [23,24]. Despite their dissimilar contacts with the hydrophobic pocket of Nef, all of these SH3-modified Hck proteins shared the wild-type Hck-like dependence on a functional R-clamp for interacting with Nef. These data are shown in Fig 7B for the Hck mutant C3 where the native RT-loop residues EAIHHE have been replaced with YSDFPW (notably containing aspartate, a charged hydrophilic residue at the SH3 position 13 instead of an isoleucine), and similar data for additional two SH3-modified Hck proteins are provided in S5 Fig

To examine the role of the Nef R-clamp in cellular system that is more relevant for HIV biology than HEK293 cells, we tested the capacity of different lentivirally to induce cellular activation in vitro differentiated THP-1-derived human macrophagic cells (Fig 8). This model for macrophage activation depends on SH3 binding by Nef, and is based on monitoring of the activation of the MAPK signaling cascade using phosphorylation of Erk1/2 as the read-out [19]. As evident from two independent experiments involving a set of YBF30 (HIV-1 N) and Nok5 (SIVcpz(P.t.s)) Nef proteins (Fig 8A) or a panel of SF2 Nef variants (Fig 8B), the capacity to activate macrophages and induce their Erk phosphorylation over the steady-state background levels required a functional R-clamp configuration.

thumbnail
Download:

Fig 8. Role of the R-clamp in activation of in vitro differentiated macrophages by Nef.

The indicated wild-type or mutant Nef proteins from HIV-1 M (panels A. and B.), HIV 1 N (panel A.), or SIVcpz(P.t.s) (panel A.) were expressed via lentiviral transduction in M1-like macrophages differentiated from THP-1 cells, followed by Western blotting analysis to compare the expression of Nef, total ERK1/2, and phosphorylated ERK1/2 in these cells or control macrophages transduced with an empty lentivector (-).

https://doi.org/10.1371/journal.ppat.1009728.g008

Finally, we also studied the role of the R-clamp in another well-established cellular function of Nef, namely enhancement of HIV-1 particle infectivity [25,26]. The underlying mechanism involving counteraction of the SERINC5 restriction factor is not SH3 dependent [27]. However, disruption of the PxxP motif has been associated with a modestly lowered capacity of Nef to increase virion infectivity [28]. When a representative panel of HIV-1 M, HIV-1 N, and SIVcpz(P.t.s) Nef alleles with wild-type or altered residues 83/120 were tested for their ability to increase the infectivity of HIV-1 particles (S6 Fig) no apparent differences were observed. This supports the idea that enhancement of HIV-1 infectivity does not depend on SH3 binding by Nef, and confirms our conclusion based on CD4 downregulation that R-clamp inactivating mutations do not universally affect Nef functionality.

Together, our results provide strong further proof to our conclusions on the 83/120 residue combinations that are required for Hck binding and activation, and demonstrate interesting structural and evolutionary plasticity in organizing a set of molecular interactions via an R-clamp principle to enable Nef to tightly bind the SH3 domain of Hck.

Discussion

Apart from the regulation of trafficking of CD4 and other membrane proteins, the majority of all cellular functions described for Nef depend on its SH3 domain binding capacity, and are lost if the highly conserved consensus SH3 ligand motif PxxPxR of Nef is mutated [7]. Here we show that Nef proteins of HIV-1 and closely related SIVs have evolved a sophisticated molecular mechanism that we have termed the R-clamp in order to coordinate their SH3 binding. This term was coined due to buttressing of the arginine residue of the Nef PxxPxR motif (R77) by the coordinated action of the Nef residues 83 and 120 together with a tryptophan residue (W37) that is conserved in almost all SH3 domains. This places R77 of Nef into close proximity with another highly conserved SH3 residue (D17) to form a salt bridge that stabilizes the Nef/SH3 complex. Thus, docking of proline-rich ligand peptides presented by native proteins, such as Nef, can involve an additional level of structural complexity for tuning of SH3 binding affinity and specificity that is not evident from studies with isolated SH3 binding peptides.

Our studies have focused on the SH3-mediated interaction between Nef and the tyrosine kinase Hck leading to enzymatic activation of Hck, which we have monitored based on induction of downstream signaling events, including paxillin phosphorylation as well as triggering of the Raf/MAPK cascade and subsequent AP-1-regulated gene expression. Nef/Hck interaction is thought to play an important role in HIV infection of cells of the myeloid lineages, such as macrophages, and is an especially tractable study system because of the high affinity of Hck SH3 domain for Nef [19]. This is due to the additional affinity brough into this interaction by tertiary contacts outside of the canonical SH3 ligand binding surface and involving the tip of the RT-loop in Hck SH3 [9,23]. Because of the lack of such RT-loop contacts, SH3 domains of other host cell proteins bind to Nef with a lower affinity resulting in more transient interactions that have remained less well characterized, but appear to include at least Lck [29], Vav [30], PACS-1 [31], Btk, and Itk [32]. Nevertheless, similar to Hck-binding, most of the other SH3 interactions by Nef are also expected to depend on formation of a salt bridge between R77 of Nef and acidic (D or E) SH3 residue 17. Thus, all such interactions, including the SH3-mediated complexes that are relevant for Nef in T lymphocytes depend on a functional R-clamp. However, it cannot be ruled out that despite containing a canonical Class II SH3 binding motif (PxxPxR) Nef (including category III) might also engage in unorthodox interactions with (an) atypical SH3 domain(s) that would not involve R-clamp coordination of R77.

Interestingly, we found that in different lentiviral Nef proteins the R-clamp has been assembled based on alternative designs, which depend on the combination of amino acids at positions 83 and 120. Moreover, the preferred 83/120 residue combinations are dissimilar in different lineages of HIV-1, SIVcpz, and SIVgor (Table 1). While the vast majority of HIV-1 group M, O and P Nefs belongs to category I, HIV-1 N Nefs are more heterogenous and fall into categories I or IV (harboring an active R-clamp) or inactive category III (Fig 9). Why would about 25% of all HIV-1 N Nefs harbor an inactive R-clamp, whereas their counterparts from HIV-1 groups M, O, and P do not? Of note, SIVcpz(P.t.t.), the simian precursor of HIV-1, comprises category I and III Nefs. Based on the phylogenic analysis of primate lentiviral Nef sequences shown in Fig 9B, we propose that the zoonotic transmission that gave rise to HIV-1 group N involved a virus carrying a Nef with a category III R-clamp.

thumbnail
Download:

Fig 9. Putative cross-species transmission and evolution of R-clamp categories.

The pie charts on the left illustrate the relative fraction of each R-clamp category in SIVcpz, SIVgor and HIV-1 groups M, N, O and P. Putative cross-species transmission events are indicated by colored arrows. The lower part of the figure shows a phylogenetic analysis of selected primate lentiviral Nefs is shown. Inferred most recent common ancestors are indicated by stars. In both images, R-clamp categories I, II, III, and IV are shown in blue, yellow, grey and orange, respectively.

https://doi.org/10.1371/journal.ppat.1009728.g009

In line with this hypothesis, as indicated in Fig 9B the inferred common ancestor of HIV-1 group N viruses belongs to category III as its Nef protein harbors a combination of Q83 and I120 [33](GenBank accession: KP059120.1). Furthermore, the SIVcpz(P.t.t.) isolate EK505 (DQ373065), a close relative of HIV-1 group N viruses [34] also expresses a category III Nef. Thus, HIV-1 group N viruses may all be the result of a category III virus, and still be adapting and evolving into categories I and IV with functional R-clamps. In this regard it should be noted that group N most likely represents the evolutionarily youngest of all HIV-1 groups [35,36], and be appreciated that the three most recently isolated group N Nefs (JN572926, MF767262, KY498771), including that of N1_FR_2011, the only group N virus isolated outside Cameroon [37], belong to categories I or IV. A single amino acid change of residue 83 (like the EK505-E83M mutant created in this study; see Fig 5) would have been sufficient to move from category III to IV, whereas the path from category III to I would require changes in both residues 83 and 120, and thus follow an evolutionary trajectory via category II. None of the currently available HIV-1 N Nef sequences fall into the R-clamp category II, but given the scarcity (n = 12) of these sequences this does not mean that such HIV-1 N Nefs could still not exist.

In contrast to HIV-1 N, group M viruses almost exclusively fall into category I, suggesting that the zoonotic jump from SIVcpz(P.t.t) to HIV-1 M involved a virus with a category I Nef R-clamp. Indeed, the inferred most recent common ancestor of HIV-1 M Nefs (KP059118.1; KP059119.1; [33] belongs to category I. This is also true for the oldest known HIV-1 M Nef (M15896) isolated in 1976 [38]. Finally, the exclusive use of category I R-clamp by Nef proteins of SIVgor, HIV-1 O, and HIV-1 P would suggest that an R-clamp category I virus might have originally been transmitted from chimpanzee to gorilla, and subsequently in two

It is of interest to note that a subtype D virus of the HIV-1 M group has been previously shown to encode a Nef protein unable to activate Hck, and that this deficiency was mapped to the unusual presence of an isoleucine residue at the Nef position 120 [39]. This study suggested that Y120 would contribute to the hydrophobic pocket of Nef that accommodates the Hck SH3 domain RT-loop, whereas I120 could not serve this function. However, these molecular contacts inferred based on our (NL4-3) Nef/SH3 X-ray structure (1EFN; [9]}) would be expected to only minor rather than critical for the Nef-Hck interaction, and a recent SH3 complex structure involving HIV-1 SF2 Nef [40] does not support such a role for Y120 at all. Moreover, our current data clearly show that category IV Nef proteins containing I120 do bind and activate Hck when paired with an appropriately matched R-clamp residue at the position 83. Thus, while the findings of Choi and Smithgall on Nef from HIV-1 ELI [39] agree with our data, they need to be interpreted in light of the R-clamp concept described here.

While the evolutionary scenarios discussed above may help to explain the occurrence of inactive category III Nefs in HIV-1 group N, it still remains unclear why about one third of all SIVcpz(Pt.t.) Nefs harbor a category III R-clamp. Furthermore, the reasons for the unique predominance of category IV Nefs in SIVcpz(P.t.s.) remain to be determined. Apart from the initial founder viruses that gave rise to the respective lentiviral lineages and species, the optimal R-clamp composition and the rate of R-clamp sequence evolution are probably determined by a complex balance between host-specific factors and sequence variation elsewhere in these viruses. To gain further insights, it would be interesting to examine a larger number of HIV-1 N Nef sequences over time and from different tissues from the same individual to understand whether the R-clamp pattern is relatively fixed or subject to rapid evolution and quasispecies variation. In the latter case, because of the highly multifunctional nature of the Nef protein, a non-functional R-clamp might provide a selective advantage in certain anatomic locations or special circumstances that might occur during or immediately after the zoonotic jump from chimpanzee to man. On the other hand, as already noted, despite the virtually universal conservation of an intact Class II SH3 binding motif PxØPxR (where Ø is a hydrophobic residue) in all Nef proteins (including R-clamp category III), it could be speculated that while unable to interact with canonical SH3 domains, R-clamp category III Nef proteins might instead show unusual specificity for some atypical SH3 proteins, which could provide them with alternative cellular functions.

In any case, R-clamp sequence variation provides a fascinating example of evolutionary plasticity of a protein interaction interface and our findings suggest that the selection pressures that have shaped Nef during primate lentiviral evolution are different depending on the HIV/SIV lineage and their host species. The dynamic capacity of Nef for altering the molecular strategy of recognizing its key host cell interaction partners, such as SH3 domains, is also important to keep in mind in attempts to develop novel HIV eradication therapies that might target the immune evasion function of Nef.

Materials and methods

Reagents and cell lines

Mouse anti-Myc (sc-40) and rabbit anti-paxillin (sc-5574) were from Santa Cruz Biotechnology. Mouse anti-GAPDH (607902) was from Biolegend). Mouse anti-pY31 paxillin was from BD Biosciences. Rabbit anti phospho-p44/42 MAPK (ERK1/2) (9101) and rabbit anti- p44/42 MAPK (ERK1/2) (9102) antibodies were from Cell Signaling Technology. Alexa Fluor 647 conjugated anti-human CD4 antibody was from SouthernBiotech. IRDye680CW goat anti-mouse IgG, IRDye800CW Streptavidin, and IRDye800CW goat anti-rabbit IgG were from LI-COR Biotechnology. HEK293, HEK293T, THP-1, and Jurkat cells were obtained from ATCC. TZM-bl cells are a HeLa-derived reporter cell line and were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH, from John C. Kappes, Xiaoyun Wu, and Tranzyme Inc. [41]. The derivation and characterization of the HEK293-based Hck-expressing HZ-1 cells have been described elsewhere [19]. HEK239T, HEK293 and HZ1 cells were grown in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Sigma) supplemented with 10% fetal bovine serum (FBS), 0.05 mg/ml penicillin, and 0.05mg/ml streptomycin. HEK293T cells were transfected in 6 well plates using a standard calcium phosphate method. Lentiviral transduction was employed to generate Jurkat cells stably expressing human CD4. Briefly, HEK293 cells were co-transfected with 2.5 μg pDelta8.9, 1.5 μg VSV-G and 3 μg pWPI-puro plasmid containing human CD4 cDNA in Opti-MEM medium with 12 μg PEI. After 5 hours, medium was refreshed with cell culture media. Supernatant was collected 48 hours post-transfection, filtered and used to infect Jurkat cells. Infected cells were selected with 6 μg/ml puromycin for 2 days. Single cell clones of CD4 transduced Jurkat cells were isolated by cellenONE X1 system. THP-1, THP-1 derived macrophages, and Jurkat cells were maintained in RPMI-1640 medium (Sigma) supplemented with 10% FBS, 0.05 mg/ml penicillin, and 0.05 mg/ml streptomycin.

Plasmids

Nef alleles from HIV-1 M SF2 (P03407), HIV-1 N 2693BA (GQ925928), HIV-1 N S4858 (KY498771), HIV-1 N YBF30 (AJ006022), HIV-1 N YBF106 (AJ271370), HIV-1 N DJO0131 (AY532635), HIV-1 O 13127k4 (AY536904), HIV-1 P RBF168 (GU111555), SIVcpz(P.t.t) CAM5 (AJ271369), SIVcpz(P.t.t) EK505 (DQ373065), SIVcpz(P.t.t) MB897 (EF535994), SIVcpz(P.t.s) ch-Nok5 (AY536915) and SIVcpz(P.t.s) Tan2 (EF394357) were cloned into the expression vector pEBB containing a C-terminal Myc-tag. Human Hck p59 cDNA (isoform b/NP_001165604) was cloned into the pEBB vector together with a 123 aa biotin acceptor domain fused to their C-termini [21]. Hck variants A1, B6, and C3 [25] were created to the same vector background. The Nef mutants (HIV-1 M SF2 Y120F, Y120I, A83E, A83E/Y120I, A83I, A83I/Y120I, A83L, A83L/Y120I, A83M, A83M/Y120I, A83Q, A83Q/Y120I, A83S, A83S/Y120I; HIV-1 N YBF30 Q83M, YBF30 I120F, 2693BA M83Q; SIVcpz(P.t.t) Cam5 I120Y, EK505 E83M, EK505 I120Y and SIVcpz (P.t.s) ch-Nok5 I83Q, ch-Nok5 I120F) were generated in the same vector backbone using standard PCR-assisted mutagenesis. All of the Nef variants mentioned above were cloned into the pWPI-GFP vector (Addgene # 12254) for lentiviral transduction of stably CD4-expressing Jurkat cells and THP-1-derived macrophages.

Immunoblots

Cells were collected and lysed on ice for 10 minutes in lysis buffer (150 mM NaCl, 50 mM Tris-HCl [pH 7.4], 1% NP-40) with protease and phosphatase inhibitors (Thermo Fisher Scientific). Cell lysates were centrifuged at 16,000 x g at 4°C for 5 min. Proteins from cell extracts were analyzed by standard SDS gel electrophoresis and Western blotting using IRDye-labeled detection reagents detailed above.

AP-1 luciferase reporter assay

HZ1 cells were transfected using TransIT-2020 reagent (Mirus) with 50 ng of Nef expression vector together with 50 ng of AP-1 pfLUC reporter plasmid [42] driving the AP-1 inducible expression of firefly luciferase, plus 50 ng of the plasmid pRL-TK (Promega) expressing low and constitutive levels of Renilla luciferase. Cells were collected and lysed with lysis buffer (Promega) on ice. A dual-luciferase reporter assay system from Promega was utilized to determine luciferase activities following the manufacturer’s protocol using Berthold Sirius single-tube luminometer detection.

Lentiviral transduction of THP-1 derived macrophages

1 x 108 THP-1 cells were infected with lentiviral vectors containing various nef alleles. 3 days post-infection, GFP positive THP-1 cells were sorted by fluorescence-activated cell sorting (FACS). Subsequently, 1 x 107 sorted THP-1 cells were seeded into each well in a 6-well plate and treated with 10 ng/ml of PMA for 2 days. Adherent cells (M0 macrophages) were further cultured for 2 days in the presence of 10 ng/ml PMA and 10 ng/mL of granulocyte-macrophage colony-stimulating factor (GM-CSF) (ThermoFisher) to differentiate them towards an M1 macrophage phenotype.

Flow cytometry analysis of CD4 downregulation

Nef-expressing HIV-1-like pseudoviruses were produced as described previously [19]. Jurkat cells stably expressing CD4 were infected with such lentiviral vectors containing various nef alleles. 3 days post-infection the cells were collected and washed twice with PBS (pH 7.4), followed by fixing with 1% Paraformaldehyde (Sigma) at room temperature for 20 min. Cells were washed twice with PBS containing 2% FBS (FACS buffer) and stained with Alexa Fluor 647 conjugated anti human CD4 antibody at room temperature for 40 min. After staining the cells were washed twice with FACS buffer and re-suspended in PBS. The CD4 cell surface expression was analyzed using a BD Accuri C6 flow cytometer (BD Biosciences) and data analysis was performed using FlowJo software (version 10.4, Ashland OR: Becton, Dickinson and Company).

Virion infectivity

To determine the effects of Nef on virion infectivity, HEK293T cells were co-transfected with pEBB expression plasmids for different Nefs or an empty vector control and an HIV-1 reporter virus lacking functional nef and vpu genes (HIV-1 NL4-3 Δnef Δvpu IRES eGFP). Two days post transfection, cell culture supernatants were harvested. To quantify infectious virus yield, 6,000 TZM-bl cells were seeded in 96-well plates and infected with the cell culture supernatant of transfected HEK293T cells in triplicate on the following day. 3 days post infection, β-galactosidase reporter gene expression was determined using the GalScreen kit (Applied Bioscience) according to the manufacturer’s instructions. In parallel, p24 concentration was determined using a home-made ELISA. Relative virion infectivity was subsequently calculated by normalizing infectious virus yield to the amount of p24.

Phylogenetic analyses

A maximum-likelihood phylogenetic tree was constructed as described previously [43]. Briefly, the 65 nucleotide sequences of the nef gene included in the analysis were aligned using MUSCLE and the phylogenetic tree was constructed using MEGA7 [44]. Most recent common ancestors (MRCA) of Nef proteins of HIV-1 groups M, O, and N had been inferred in a previous study [33].

Supporting information

S4 Fig. One-hundred ns all-atom molecular dynamics simulations show that stabilization of R77 side chain provided by the R-clamp in the wild-type complex could be reproduced by Met, Ile and Trp in the A83M/Y120I double mutant.

Analysis of the MD trajectories show that in the wild type complex the stacked Tyr-Arg-Trp π-cation-π interaction remains stable, as interpreted from Arg to Tyr and Arg to Trp side chain distances, which show little fluctuations around their average values, 4.2 Å (A) and 3.5 Å (B). In (C) is shown a snapshot from the wild type complex simulation in which these distances are close to their average values. In the double mutant the Arg to Trp distance is on the average about 0.5 Å longer than that in the wild type complex (E), but also remains stable around its average value, 4.0 Å. If 6 Å is taken as the maximum distance for a cation-π interaction [47] the observed distances are well within the limit. Likewise, hydrophobic contacts to and between Met and Ile on the other side of the Arg plane remain relatively stable. Non-bonded contacts are likely to be bolstered by Met sulfur [48]. Ile and Arg sidechains are on average 4.4 Å apart (D). Met to Arg (F) and Ile to Met (G) distances show more variation, but are for the majority of time close to about 4.6 and 3.9 Å. In (H) is shown a snapshot from the double mutant complex simulation in which these distances are close to their average values. A salt bridge between Arg and Asp side chains is present in 96% (WT) and 93% (double mutant) of the simulation frames. MD simulations in explicit solvent were performed with AMBER 20 [24] using the ff14SB force field. The WT and A83M/Y120I complexes were placed in a cubic box with a minimum solute-box distance of 10 Å, and solvated with TIP3P water molecules. Six sodium ions were added to neutralize the system. After minimization, heating and equilibration of the system, the production 100-ns MD simulations were performed with periodic boundary conditions at 300 K. The temperature was maintained by using the Langevin thermostat, whereas the pressure was kept at 1 bar using the Berendsen barostat [49]. The time step was set to 2 fs. Long-range electrostatic interactions were treated using the Particle Mesh Ewald method [50] with a cut-off of 10 Å. Bond lengths involving hydrogen atoms were constrained by SHAKE [51]. Analyses of the trajectories were carried out with CPPTRAJ [52].

https://doi.org/10.1371/journal.ppat.1009728.s004

(PDF)

S5 Fig. Role of R-clamp residues 83/120 in intracellular Hck-Nef complex formation.

HEK293 cells were transfected with Myc-tagged wild-type HIV-1 M SF2 Nef (WT) or its mutants including Y120I, A83L-Y120I, P76A+P78A (AxxA), A83Q, and A83Q-Y120I) together with biotin acceptor domain-tagged SH3 mutated Hck-p59 variants Hck-A1 (A.) or Hck-B6 (B.). Similarly, YBF30 or its R-clamp gain-of-function mutant Q83M were co-expressed with biotin acceptor domain-tagged wild-type Hck-p59 or its SH3 mutant Hck-B6. (C.). The Nef-contacting RT-loop residues centered around the critical Hck SH3 amino acid 13 (indicated in bold) are shown on top of these Figs. Lysates of the transfected cells were subjected to anti-Myc immunoprecipitation followed by Western blotting analysis of the immune complexes using labeled streptavidin (top panels). Equal Hck and Nef expression in the total lysates was confirmed by labeled streptavidin (Hck) (middle panels) or an anti-Myc (Nef) antibody (bottom panels).

https://doi.org/10.1371/journal.ppat.1009728.s005

(PDF)

S6 Fig. Role of the R-clamp in Nef-mediated enhancement of virion infectivity.

(A) HEK293T cells were co-transfected with pEBB expression plasmids for the indicated Nefs and an HIV-1 reporter virus lacking functional nef and vpu genes (HIV-1 NL4-3 Δnef Δvpu IRES eGFP). Two days post transfection cell culture supernatants were harvested. Infectious virus yield was determined by infecting TZM-bl reporter cells and normalized to the amount of p24 (as determined by ELISA) to calculate virion infectivity. Mean values +/- SEM of four independent experiments are shown. (B) Two days post transfection cells were harvested for Western Blot analysis. Nef was detected via an anti-myc tag antibody. GAPDH served as loading control.

https://doi.org/10.1371/journal.ppat.1009728.s006

(PDF)

Acknowledgments

We thank Ms. Virpi Syvälahti for expert technical assistance.

References

  1. 1. Sauter D, Kirchhoff F. Key viral adaptations preceding the AIDS pandemic. Cell Host Microbe. 2019;25(1):27–38. Epub 2019/01/11. pmid:30629915.
  2. 2. Désiré N, Cerutti L, Le Hingrat Q, Perrier M, Emler S, Calvez V, et al. Characterization update of HIV-1 M subtypes diversity and proposal for subtypes A and D sub-subtypes reclassification. Retrovirology. 2018;15(1):80. Epub 2018/12/24. pmid:30577842.
  3. 3. Robertson DL, Anderson JP, Bradac JA, Carr JK, Foley B, Funkhouser RK, et al. HIV-1 nomenclature proposal. Science. 2000;288(5463):55–6. Epub 2000/04/15. pmid:10766634.
  4. 4. Yamaguchi J, Vallari A, McArthur C, Sthreshley L, Cloherty GA, Berg MG, et al. Brief Report: Complete Genome Sequence of CG-0018a-01 Establishes HIV-1 Subtype L. J Acquir Immune Defic Syndr. 2020;83(3):319–22. Epub 2019/11/07. pmid:31693506.
  5. 5. Kirchhoff F, Schindler M, Specht A, Arhel N, Munch J. Role of Nef in primate lentiviral immunopathogenesis. Cell Mol Life Sci. 2008;65(17):2621–36. Epub 2008/04/29. pmid:18438604.
  6. 6. Arhel NJ, Kirchhoff F. Implications of Nef: host cell interactions in viral persistence and progression to AIDS. Curr Top Microbiol Immunol. 2009;339:147–75. Epub 2009/12/17. pmid:20012528.
  7. 7. Saksela K. Interactions of the HIV/SIV pathogenicity factor Nef with SH3 domain-containing host cell proteins. Curr HIV Res. 2011;9(7):531–42. Epub 2011/11/23. pmid:22103837.
  8. 8. Staudt RP, Alvarado JJ, Emert-Sedlak LA, Shi H, Shu ST, Wales TE, et al. Structure, function, and inhibitor targeting of HIV-1 Nef-effector kinase complexes. J Biol Chem. 2020;295(44):15158–71. Epub 2020/08/31. pmid:32862141.
  9. 9. Lee CH, Saksela K, Mirza UA, Chait BT, Kuriyan J. Crystal structure of the conserved core of HIV-1 Nef complexed with a Src family SH3 domain. Cell. 1996;85(6):931–42. Epub 1996/06/14. pmid:8681387.
  10. 10. Moarefi I, LaFevre-Bernt M, Sicheri F, Huse M, Lee CH, Kuriyan J, et al. Activation of the Src-family tyrosine kinase Hck by SH3 domain displacement. Nature. 1997;385(6617):650–3. Epub 1997/02/13. pmid:9024665.
  11. 11. Guiet R, Poincloux R, Castandet J, Marois L, Labrousse A, Le Cabec V, et al. Hematopoietic cell kinase (Hck) isoforms and phagocyte duties—from signaling and actin reorganization to migration and phagocytosis. Eur J Cell Biol. 2008;87(8–9):527–42. Epub 2008/06/10. pmid:18538446.
  12. 12. Kumar A, Herbein G. The macrophage: a therapeutic target in HIV-1 infection. Mol Cell Ther. 2014;2:10. Epub 2014/01/01. pmid:26056579.
  13. 13. Greenberg ME, Iafrate AJ, Skowronski J. The SH3 domain-binding surface and an acidic motif in HIV-1 Nef regulate trafficking of class I MHC complexes. Embo j. 1998;17(10):2777–89. Epub 1998/06/10. pmid:9582271.
  14. 14. Imle A, Abraham L, Tsopoulidis N, Hoflack B, Saksela K, Fackler OT. Association with PAK2 Enables Functional Interactions of Lentiviral Nef Proteins with the Exocyst Complex. MBio. 2015;6(5):e01309–15. pmid:26350970.
  15. 15. Rudolph JM, Eickel N, Haller C, Schindler M, Fackler OT. Inhibition of T-cell receptor-induced actin remodeling and relocalization of Lck are evolutionarily conserved activities of lentiviral Nef proteins. J Virol. 2009;83(22):11528–39. Epub 2009/09/04. pmid:19726522.
  16. 16. Saksela K, Cheng G, Baltimore D. Proline-rich (PxxP) motifs in HIV-1 Nef bind to SH3 domains of a subset of Src kinases and are required for the enhanced growth of Nef+ viruses but not for down-regulation of CD4. EMBO J. 1995;14(3):484–91. Epub 1995/02/01. pmid:7859737.
  17. 17. Mayer BJ. The discovery of modular binding domains: building blocks of cell signalling. Nat Rev Mol Cell Biol. 2015;16(11):691–8. Epub 2015/10/01. pmid:26420231.
  18. 18. Saksela K, Permi P. SH3 domain ligand binding: What’s the consensus and where’s the specificity? FEBS Lett. 2012;586(17):2609–14. Epub 2012/06/20. pmid:22710157.
  19. 19. Zhao Z, Fagerlund R, Baur AS, Saksela K. HIV-1 Nef-induced secretion of the proinflammatory protease TACE into extracellularvesicles is mediated by Raf-1, and can be suppressed by clinical protein kinase inhibitors. J Virol. 2021. Epub 2021/02/19. pmid:33597213.
  20. 20. Collette Y, Arold S, Picard C, Janvier K, Benichou S, Benarous R, et al. HIV-2 and SIV nef proteins target different Src family SH3 domains than does HIV-1 Nef because of a triple amino acid substitution. J Biol Chem. 2000;275(6):4171–6. Epub 2000/02/08. pmid:10660579.
  21. 21. Zhao Z, Kesti T, Ugurlu H, Baur AS, Fagerlund R, Saksela K. Tyrosine phosphorylation directs TACE into extracellular vesicles via unconventional secretion. Traffic. 2019;20(3):202–12. Epub 2018/12/21. pmid:30569492.
  22. 22. Kirchhoff F, Schindler M, Bailer N, Renkema GH, Saksela K, Knoop V, et al. Nef proteins from simian immunodeficiency virus-infected chimpanzees interact with p21-activated kinase 2 and modulate cell surface expression of various human receptors. J Virol. 2004;78(13):6864–74. Epub 2004/06/15. pmid:15194762.
  23. 23. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, et al. UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem. 2004;25(13):1605–12. Epub 2004/07/21. pmid:15264254.
  24. 24. Case DA, Belfon K, Ben-Shalom IY, Brozell SR, Cerutti DS, Cheatham TEI, et al. AMBER 2020: University of California, San Francisco; 2020.
  25. 25. Hiipakka M, Poikonen K, Saksela K. SH3 domains with high affinity and engineered ligand specificities targeted to HIV-1 Nef. J Mol Biol. 1999;293(5):1097–106. Epub 1999/11/05. pmid:10547288.
  26. 26. Horenkamp FA, Breuer S, Schulte A, Lulf S, Weyand M, Saksela K, et al. Conformation of the dileucine-based sorting motif in HIV-1 Nef revealed by intermolecular domain assembly. Traffic. 2011;12(7):867–77. Epub 2011/04/12. pmid:21477083.
  27. 27. Chowers MY, Spina CA, Kwoh TJ, Fitch NJ, Richman DD, Guatelli JC. Optimal infectivity in vitro of human immunodeficiency virus type 1 requires an intact nef gene. J Virol. 1994;68(5):2906–14. Epub 1994/05/01. pmid:8151761.
  28. 28. Firrito C, Bertelli C, Vanzo T, Chande A, Pizzato M. SERINC5 as a New Restriction Factor for Human Immunodeficiency Virus and Murine Leukemia Virus. Annu Rev Virol. 2018;5(1):323–40. Epub 2018/09/29. pmid:30265629.
  29. 29. Shi J, Xiong R, Zhou T, Su P, Zhang X, Qiu X, et al. HIV-1 Nef antagonizes SERINC5 restriction by downregulation of SERINC5 via the endosome/lysosome system. J Virol. 2018;92(11). Epub 2018/03/09. pmid:29514909.
  30. 30. Fackler OT, Moris A, Tibroni N, Giese SI, Glass B, Schwartz O, et al. Functional characterization of HIV-1 Nef mutants in the context of viral infection. Virology. 2006;351(2):322–39. Epub 2006/05/11. pmid:16684552.
  31. 31. Collette Y, Dutartre H, Benziane A, Ramos M, Benarous R, Harris M, et al. Physical and functional interaction of Nef with Lck. HIV-1 Nef-induced T-cell signaling defects. J Biol Chem. 1996;271(11):6333–41. Epub 1996/03/15. pmid:8626429.
  32. 32. Rauch S, Pulkkinen K, Saksela K, Fackler OT. Human immunodeficiency virus type 1 Nef recruits the guanine exchange factor Vav1 via an unexpected interface into plasma membrane microdomains for association with p21-activated kinase 2 activity. J Virol. 2008;82(6):2918–29. Epub 2007/12/21. pmid:18094167.
  33. 33. Blagoveshchenskaya AD, Thomas L, Feliciangeli SF, Hung CH, Thomas G. HIV-1 Nef downregulates MHC-I by a PACS-1- and PI3K-regulated ARF6 endocytic pathway. Cell. 2002;111(6):853–66. Epub 2003/01/16. pmid:12526811.
  34. 34. Tarafdar S, Poe JA, Smithgall TE. The accessory factor Nef links HIV-1 to Tec/Btk kinases in an Src homology 3 domain-dependent manner. J Biol Chem. 2014;289(22):15718–28. Epub 2014/04/12. pmid:24722985.
  35. 35. Kluge SF, Mack K, Iyer SS, Pujol FM, Heigele A, Learn GH, et al. Nef proteins of epidemic HIV-1 group O strains antagonize human tetherin. Cell Host Microbe. 2014;16(5):639–50. Epub 2014/12/20. pmid:25525794.
  36. 36. Bibollet-Ruche F, Heigele A, Keele BF, Easlick JL, Decker JM, Takehisa J, et al. Efficient SIVcpz replication in human lymphoid tissue requires viral matrix protein adaptation. J Clin Invest. 2012;122(5):1644–52. Epub 2012/04/17. pmid:22505456.
  37. 37. Sauter D, Hué S, Petit SJ, Plantier JC, Towers GJ, Kirchhoff F, et al. HIV-1 Group P is unable to antagonize human tetherin by Vpu, Env or Nef. Retrovirology. 2011;8:103. Epub 2011/12/17. pmid:22171785.
  38. 38. Wertheim JO, Worobey M. Dating the age of the SIV lineages that gave rise to HIV-1 and HIV-2. PLoS Comput Biol. 2009;5(5):e1000377. Epub 2009/05/05. pmid:19412344.
  39. 39. Delaugerre C, De Oliveira F, Lascoux-Combe C, Plantier JC, Simon F. HIV-1 group N: travelling beyond Cameroon. Lancet. 2011;378(9806):1894. Epub 2011/11/29. pmid:22118443.
  40. 40. Srinivasan A, York D, Butler Jr., Jannoun-Nasr R, Getchell J, McCormick J, et al. Molecular characterization of HIV-1 isolated from a serum collected in 1976: nucleotide sequence comparison to recent isolates and generation of hybrid HIV. AIDS Res Hum Retroviruses. 1989;5(2):121–9. Epub 1989/04/01. pmid:2713163.
  41. 41. Choi HJ, Smithgall TE. Conserved residues in the HIV-1 Nef hydrophobic pocket are essential for recruitment and activation of the Hck tyrosine kinase. J Mol Biol. 2004;343(5):1255–68. Epub 2004/10/20. pmid:15491611.
  42. 42. Aldehaiman A, Momin AA, Restouin A, Wang L, Shi X, Aljedani S, et al. Synergy and allostery in ligand binding by HIV-1 Nef. Biochem J. 2021;478(8):1525–45. Epub 2021/04/01. pmid:33787846.
  43. 43. Platt EJ, Wehrly K, Kuhmann SE, Chesebro B, Kabat D. Effects of CCR5 and CD4 cell surface concentrations on infections by macrophagetropic isolates of human immunodeficiency virus type 1. J Virol. 1998;72(4):2855–64. Epub 1998/04/03. pmid:9525605.
  44. 44. Pesu M, Takaluoma K, Aittomaki S, Lagerstedt A, Saksela K, Kovanen PE, et al. Interleukin-4-induced transcriptional activation by stat6 involves multiple serine/threonine kinase pathways and serine phosphorylation of stat6. Blood. 2000;95(2):494–502. Epub 2000/01/11. pmid:10627454.
  45. 45. Nakano Y, Yamamoto K, Ueda MT, Soper A, Konno Y, Kimura I, et al. A role for gorilla APOBEC3G in shaping lentivirus evolution including transmission to humans. PLoS Pathog. 2020;16(9):e1008812. Epub 2020/09/12. pmid:32913367.
  46. 46. Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016;33(7):1870–4. Epub 2016/03/24. pmid:27004904.
  47. 47. Gallivan JP, Dougherty DA. Cation-pi interactions in structural biology. Proc Natl Acad Sci U S A. 1999;96(17):9459–64. Epub 1999/08/18. pmid:10449714.
  48. 48. Gómez-Tamayo JC, Cordomí A, Olivella M, Mayol E, Fourmy D, Pardo L. Analysis of the interactions of sulfur-containing amino acids in membrane proteins. Protein Sci. 2016;25(8):1517–24. Epub 2016/05/31. pmid:27240306.
  49. 49. Berendsen HJC, Postma JPM, van Gunsteren WF, DiNola A, Haak JR. Molecular dynamics with coupling to an external bath. J Chem Phys. 1984;81:3684–90.
  50. 50. Darden T, York D, Pedersen L. Particle mesh Ewald: an N·log(N) method for Ewald sums in large systems. J Chem Phys. 1993;98:10089–92.
  51. 51. Ryckaert J-P, Ciccotti G, Berendsen HJC. Numerical integration of the cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes. J Comput Phys. 1977;23:327–41.
  52. 52. Roe DR, Cheatham TE 3rd. PTRAJ and CPPTRAJ: Software for Processing and Analysis of Molecular Dynamics Trajectory Data. J Chem Theory Comput. 2013;9(7):3084–95. Epub 2013/07/09. pmid:26583988.
Check for updates via CrossMark

Subject Areas

?

For more information about PLOS Subject Areas, click here.

We want your feedback.Do these Subject Areas make sense for this article? Click the target next to the incorrect Subject Area and let us know. Thanks for your help!

  • HIV-1 
  • Transfection 
  • Viral evolution 
  • Chimpanzees 
  • Macrophages 
  • Primates 
  • SIV 
  • Luciferase 
Источник: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1009728

1 Replies to “Acid properties - Crack Key For U”

Leave a Reply

Your email address will not be published. Required fields are marked *