Biochemistry Exam 1

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Comparing H2O to H2S, why does water have such a comparatively HIGH boiling point compared to H2S? A) H2O possesses 2 lone pairs of non-bonding electrons and H2S does not B) H2S has a great molecular weight and makes fewer van der Waals interactions C) 3D structure of H2O is significantly different from that of H2S D) The oxygen in H2O is more electronegative than sulfur and is therefore better at H-bonding E) The sulfur in H2S is more electronegative than oxygen and is therefore better at H-bonding

D) The oxygen in H2O is more electronegative than sulfur and is therefore better at H-bonding

What is the predominant type of IMF in CHF3?

Dipole-dipole

What is the predominant type of IMF in OF2?

Dipole-dipole

How does entropy dictate the solubility/insolubility of molecules and gases?

Entropy dictates the solubility/insolubility of molecules and gases, because when hydrophilic molecules dissolve, because they do not form bonds with water and water does not form hydration shells around them, water forms a lattice-like structure around the nonpolar molecules. This lattice is more highly ordered than the rest of the water, and its formation requires free-energy, so it will never spontaneously happen. The free energy is minimized when hydrophobic molecules cluster together, which is why they will not be soluble in water

When amphiphilic solutes are put into water, where is entropy increasing, and where is entropy decreasing?

Entropy is decreasing for micelle and/or vesicle formation because these are highly ordered structures. Entropy is increasing on the outside of the micelles and vesicles, where water molecules can interact with themselves freely, and/or the polar heads of the amphipathic molecules.

Why do micelles and/or vesicles form spontaneously in vivo?

Even though the formation of the initial micelle is entropically unfavorable, the overall process is favorable because: 1. Heat of formation because the formation of hydrogen bonds between the water molecules and the polar side of the bilayer, and if we make an interaction, we get a little bit of energy back out 2. As the water molecules are squeezed out of the micelles, the water molecules do not have to form cages around the hydrocarbon tails and are free to interact with other water molecules, the polar head groups, etc. and can exist in many more conformations 3. In addition, the amphipathic molecules are free to mix and mingle with one another

What is the predominant type of IMF in HF?

Hydrogen bonding

How do hydrophobic side chains interact in a folded protein?

Hydrophobic side chains interact with each other via weak van der Waals interactions

Why, when we dump a hydrocarbon solution into an aqueous solution, will we end up with two distinct phases?

If we did not end up with two distinct phases, an aqueous phase and an organic phase, then the water molecules would have to become more ordered to put the solute into solution, which is entropically unfavorable, but if we get two distinct phases, within each respective phase the molecules are free to move around, which is entropically favorable. It is more entropically favorable for the water to form one cage around the giant hydrocarbon solution than to form multiple cages around multiple hydrocarbon molecules

When water interacts with nonpolar solutes, does overall entropy increase or decrease for the process and why?

It decreases because as the water molecules are aggregating around the non polar solutes they are having to do it in a very structured way such that their nonpolar face is towards the nonpolar molecule

What is a hydrogen bond donor?

It is the atom to which hydrogen is covalently bound to

What is glycogen?

It is used for short term energy and it is a repeat polymer of glucose

What type of energy do lipids provide and why is this?

Long term energy because they are very reduced, and the more reduced they are, the more energy you can get out of them

Why are Van der Waals forces critical for micelle formation?

Micelles are formed by amphipathic molecules, that have a hydrophilic head and a hydrophobic hydrocarbon tail. Van der Waals forces are critical for micelle formation, because they forces interact when the hydrocarbon tails are parallel, which happens when they form a monolayer. The close packing of the hydrocarbon side chains is due to the van der Waals interactions between them

The hydrides of group 5A are NH3, PH3, AsH3, and SbH3. Arrange them from highest to lowest boiling point.

NH3, SbH3, AsH3, PH3

With respect to the formation of micelles and/or lipid bilayers, is the change in entropy positive or negative?

Negative, because we are going from disorder, to a very ordered situation

The side chain of histidine has a typical pKa value in the range of 6.5-7.4. However, when analyzing the pKa values in a particular protein, scientists determined that one particular His residue has an unusually low pKa value of 4.8. Which of the following statements correctly explain this anomaly? (choose all that apply) 1) The microenvironment around a residue can impact its pKa value. 2) A positively charged amino acid must be in close proximity to this residue. 3) A negatively charged amino acid must be in close proximity to this residue. 4) This residue must be located on the surface of the protein.

Only 1 and 2 (the presence of a nearby positive charge is likely to decrease the pKa of the His side chain because two neighboring positive charges will repel each other. There is not enough information to draw any conclusion about the location of this residue within the protein structure)

When water interacts with polar solutes, does overall entropy for the process increase or decrease, and why?

Overall entropy increases, because introducing a polar solute provides additional conformations that water can exist. If there is just water, water can make and break interactions with many different water molecules, but when we add 1M NaCl, we have Na+ ions and Cl- ions, so now water can exist in more conformational states because now it can also interact with Na+ ions and Cl- ions, so it increases entropy.

Why is the hydrogen of a water molecule partially positive?

Oxygen has a high electronegativity, so the electrons in the H-O bond are close to oxygen than they are to hydrogen

'It is possible to make a buffer that functions well near pH 7 using citric acid, which contains only carboxylate groups. Why?

Successive deprotonations increase the negative charge density on the resulting anion, resulting in unfavorable electrostatic repulsions between the carboxylate anions, which reduces the likelihood that a proton would dissociate. It is more favorable for the proton to remain bound to reduce unfavorable charge repulsions

What is ∆E˚?

The change in the standard reduction potential.

What is ∆H?

The heat that is given off in a reaction

What is pI?

The isoelectric point, which is where the pH of a solution when the net charge on an ampholyte is 0

Why is the bond angle in a water molecule not 109.5˚?

The lone pairs in the non-bonding electrons take up more 3D space than bonding electrons, and make it so the bond angle must be less than 109.5˚

What is the role of entropy in micelle formation?

The role of entropy in the process of micelle formation is that in the formation of a micelle, the hydrophobic tails all cluster together in order to limit the contact with the surrounding water molecules (which would cause the water molecules to form the unfavorable and more organized lattices), and the hydrophilic heads are in contact with the water

What gives the alpha helix the regular, repeating pattern?

The similarity of all of the bond angles that are sterically allowed according to the Ramachandran plot

How are the electron pairing compounds in the electron transport chain arranged?

They are arranged based on standard reduction potential so that electrons flow from one electron pairing compound to another based on the affinity of one electron pairing substance to another. They will flow from a lower standard reduction potential compound to one with a higher standard reduction potential

At what point on a titration curve do we have the greatest buffering capacity?

When the pH approaches pKa. (this is because when we use the Henderson-Hasselbach equation, when pH=pKa, the ratio of [A-]:[HA] is 1, meaning that or [HA]:[A-] is 1, so there is the max amount of acid to neutralize any base, and base to neutralize any acid, so the pH will change minimally here)

The horizontal part of the titration curve represents what?

Where pH=pKa and the buffering region (this is because HA and A- are in equal concentrations, so if the pH changes HA can either release a proton or A- can accept a proton at the best ability at this zone)

What does a nucleotide consist of?

a base, a sugar, and a phosphate group

What is an exergonic reaction?

a reaction that releases free energy (∆G < 0) and is thermodynamically favored

What is an endergonic reaction?

a reaction that required free energy to be taken up from the surroundings (∆G > 0) and is not energetically favorable

Which are true of the correct conditions for forming hydrogen bonds? a) A hydrogen bond is possible with only certain hydrogen-containing compounds. b) A hydrogen atom acquires a partial positive charge when it is covalently bonded to an F atom. c) The CH4 molecule exhibits hydrogen bonding. d) Hydrogen bonding occurs when a hydrogen atom is covalently bonded to an N, O, or F atom. e) A hydrogen bond is equivalent to a covalent bond.

a) A hydrogen bond is possible with only certain hydrogen-containing compounds. b) A hydrogen atom acquires a partial positive charge when it is covalently bonded to an F atom. d) Hydrogen bonding occurs when a hydrogen atom is covalently bonded to an N, O, or F atom.

Much is made of water's ability to dissolve a wide variety of molecules and yet, one of its most important characteristics is its effect on nonpolar, uncharged molecules. Which of the following statements is NOT true about this effect? a) Hydrophobic molecules are driven together largely because of forces of attraction between them. b) Water molecules form clathrates or cages that surround nonpolar molecules or particles. c) The hydrophobic effect plays an important role in the folding of proteins and the assembly of lipid bilayers. d) All of the listed statements are true.

a) Hydrophobic molecules are driven together largely because of forces of attraction between them.

What does it mean to reduce a molecule?

add a hydrogen or remove an oxygen

In biochemistry, most reactions take place in what type of solution?

aqueous

Noncovalent bonds are critically important to the function of biomolecules. Which of the following is/are (a) valid reason(s) for this importance? a) The effect of these weak interactions is cumulative. b) Noncovalent interactions are made up of hydrogen bonding, electrostatic interactions, and dispersion forces. c) Their bond energies are ~10-100 times weaker than ordinary covalent bonds. d) All of the listed statements are valid reasons for this importance.

d) All of the listed statements are valid reasons for this importance.

How does vapor pressure change as the strength of intermolecular forces increase?

decreases (strong intermolecular forces lead to less evaporation, which means fewer molecules will be in the gaseous state and with fewer in that state, the pressure exerted by the gas will be lower)

Why is liquid water entropically favorable?

each water molecule can exist in many other conformational states and interact with other molecules because they are rapidly making and breaking their hydrogen bonds, and entropy is a measure of the number of conformational states that a molecule can exist in

Substances with weak intermolecular forces tend to be in what state at room temperature?

gas

Glycogen is a repeat polymer of what monomer?

glucose

All amino acids have a chiral α-carbon EXCEPT ________.

glycine (it has two hydrogens attached to the α-carbon)

How does water interact with the polar heads of phospholipids?

hydrogen bonds

Which forces are important in stabilizing the DNA double helix?

hydrogen bonds between complementary base pairs inside the helix and the Van der Waals base-stacking interaction.

How does the surface tension change as the strength of intermolecular forces increase?

increase

How does the boiling point change as the strength of intermolecular forces increase?

increases

How does viscosity change as the strength of intermolecular forces increase?

increases

Classify this intermolecular force as moderate or weak: ion-dipole

moderate (they have a permanent full charge)

Classify this intermolecular force as moderate or weak: hydrogen bonding

moderate (they have an especially strong dipole force)

What is an amphiphilic/amphipathic molecule?

molecules that possess a polar and nonpolar region

What is the monomeric unit of a carbohydrate?

monosaccharides

A spontaneous reaction will have what value of ∆G?

negative

What is the ultimate electron acceptor in the electron transport chain?

oxygen (which makes sense, because oxygen has a very high reduction potential, and so therefore it is thermodynamically favorable for electrons to flow to oxygen in the ETC because of ∆G=-nF∆E)

A non-spontaneous reaction will have what value of ∆G?

positive

What is Ka?

the amount a weak acid that dissociates in solution

What is entropy?

the degree of disorder or randomness in a system and the number of conformational states that a molecule can exist in

What is Gibbs free energy?

the energy that is available to do work, and it determines whether a process is thermodynamically favored at a given temperature

Why are phospholipids in a micelle or lipid bilayer free to "mix and mingle" with each other?

the hydrocarbon tails form van der Waals interactions with each other, and these are easy to break and reform. In addition, the heads are hydrogen bonded to water molecules, and to themselves, and these bonds are also relatively easy to break

What is the standard reduction potential?

the measure of the tendency of a chemical species to acquire electrons (H atoms) and be reduced

What is pKa?

the negative log of Ka, which is the amount a weak acid that dissociates in solution

What type of interaction do the hydrocarbon tails of phospholipids form?

they form van der Waals interactions between themselves

What type of energy do carbohydrates provide and why is this?

they provide short term energy since they are not very reduced, and reduced compounds store more energy

Classify this intermolecular force as moderate or weak: dipole-dipole

weak (they have a permanent partial charge)

Classify this intermolecular force as moderate or weak: London dispersion

weak (they involve temporary partial charges)

What type of interactions do the hydrocarbon tails of amphipathic molecules make with each other?

weak van der Waals interactions that break easily, allowing them to be fluid tit

Which of the following characteristics are true about a typical peptide (amide) bond? 1) The bond is planar. 2) There is free rotation about the carbonyl carbon and nitrogen bond. 3) There is substantial double-bond character to this bond. 4) There is a net negative charge on nitrogen and net positive charge on oxygen.

1 and 3 (the carbonyl carbon-nitrogen bond is the bond that has a partial double bond)

Which statements about lipid bilayers are true? 1) Their formation is driven by the hydrophobic effect. 2) The amphipathic molecules that make up the bilayers orient themselves such that their polar head groups point out toward the aqueous solvent and their hydrophobic tails are pointed toward the interior of the bilayer. 3) They serve the role of physical compartmentalization of components within a cell. 4) They are made primarily from phospholipids.

1) Their formation is driven by the hydrophobic effect. 2) The amphipathic molecules that make up the bilayers orient themselves such that their polar head groups point out toward the aqueous solvent and their hydrophobic tails are pointed toward the interior of the bilayer. 3) They serve the role of physical compartmentalization of components within a cell. 4) They are made primarily from phospholipids.

The activity of an enzyme requires a glutamic acid to display its −COOH functional group in the protonated state. Suppose the pKa of the −COOH group is 4.07. 1. Will the enzyme be more active at pH 3.5 or 4.5? Explain. 2. What fraction of the enzymes will be active at pH=4.07? Explain. 3. At what pH will the enzyme show 78% of maximal activity?

1. The enzyme will be more active at pH of 3.5, because protonation is favored when pH < pKa (think Henderson-Hasselbalch Equation) 2. when pH=pKa, the ionizable group is 50% protonated, so 50% of the enzymes will be in the active state 3. 3.52

What are the structural and physical properties that make water ideal as a solvent for life? List a few characteristics.

1. The oxygen molecule in water has 2 sets of unpaired electrons, that make it perfect for forming hydrogen bonds between molecules 2. Water is a very polar molecule, and has a high dielectric constant, and as a solvent, it will reduce the electrostatic force between two ions, and make it harder for them to get back together 3. Water is an excellent solvent for ionic compounds, because the interactions of the negative ends of water dipoles with cations and the interactions of the positive ends of water dipoles with anions causes ions in a water solution to become hydrated, and surrounded by hydration shells of water 4. Water can also disrupt molecules that contain internal structural hydrogen bonds, and unfold them 5. Non-ionic, but polar organic molecules can also dissolve in water because the interaction of their dipole with water's strong dipole promotes solubility

What fraction of a ribosome consists of protein, and what fraction consists of rRNA?

1/3 protein and 2/3 rRNA

What is the bond angle in a water molecule?

104.5˚

What fraction of our RNA is structural, and what fraction is mRNA?

2/3 structural, 1/3 mRNA

Consider a small protein containing 101 amino acid resides. The protein will have 200 bonds around which rotation can occur. Assume that three orientations are possible fore ach bond. How many random coil conformations will be possible for this protein?

3^200=2.7 x 10^95

How many other water molecules can one water molecule simultaneously hydrogen bond with?

Which of the following molecules can exhibit hydrogen bonding as a pure liquid? A) CF2H2 B) N2H4 C) H2O D) ClBr4 E) C6H5COH F)COOHCOOH G) HCl

B) N2H4 C) H2O F)COOHCOOH

Why do we use a lot of weak bonds in the formation of macromolecular structures instead of the stronger covalent bonds?

Because if we use a lot of weaker non covalent interactions, each interaction can provide great stability, but the ease at which each individual non covalent bond can be broken and reformed gives the structures the dynamic flexibility necessary for their function

What makes it so that 6 feet of DNA is able to be folded up and fit in just one epithelial cell?

Because nucleic acids are held together with non covalent interactions that are broken with relative ease, thus giving them great flexibility to be folded up. If they were held together with rigid, great covalent bonds, they would not be this flexible and able to do that

What are the N-terminal and C-terminal residues for the following peptide sequence? Peptide sequence: EASY A) Glutamic acid is the N-terminal residue; tryptophan is the C-terminal residue. B) Y is the N-terminal residue; E is the C-terminal residue. C) E is the N-terminal residue; Y is the C-terminal residue. D) Aspartic acid is the N-terminal residue; tyrosine is the C-terminal residue.

C) E is the N-terminal residue; Y is the C-terminal residue.

Which of the following is FALSE when considering van der Waals interactions? A) Van der Waals radii can determine molecular surfaces. B) Molecules that interact by van der Waals forces do not interpenetrate. C) They are not important in determining the stability of three-dimensional structures of proteins. D) The total interaction energy is the sum of the attractive and repulsive forces.

C) They are not important in determining the stability of three-dimensional structures of proteins.

Rank in order of decreasing vapor pressure: CH3CH2CH2CH2OH, CH4, CH3CH(CH3)CH3CH3, CH3CH2CH2CH2CH3

CH4, CH3CH(CH3)CH2CH3, CH3CH3CH3CH3CH3, CH3CH2CH2CH2OH

Rank the following substances in order from most soluble in water to least soluble in water: C3H8, CH3OH, CuSO4, C4H10

CuSO4, CH3OH, C4H10, C3H8 (In this order, the presence of ions, the presence of a dipole moment, and the presence of London dispersion forces, favor hydrophilicity)

One method for separating polypeptides makes use of their different solubilities. The solubility of large polypeptides in water depends upon the relative polarity of their R groups, particularly on the number of ionized groups: the more ionized groups there are, the more soluble the polypeptide. Which of each pair of the polypeptides that follow is more soluble at the indicated pH? (a) (Gly)20 or (Glu)20 at pH 7.0 (b) (Lys-Ala)3 or (Phe-Met)3 at pH 7.0 (c) (Ala-Ser-Gly)5 or (Asn-Ser-His)5 at pH 6.0 (d) (Ala-Asp-Gly)5 or (Asn-Ser-His)5 at pH 3.0

(a) (Glu)20; it is highly negatively charged (polar) at pH 7. (Gly)20 is uncharged except for the amino- and carboxyl-terminal groups. (b) (Lys-Ala)3; this is highly positively charged (polar) at pH 7. (Phe-Met)3 is much less polar and hence less soluble. (c) (Asn-Ser-His)5; both polymers have polar Ser side chains, but (Asn-Ser-His)5 also has the polar Asn side chains and partially protonated His side chains. (d) (Asn-Ser-His)5; at pH 3, the carboxylate groups of Asp residues are partially protonated and neutral, whereas the imidazole groups of His residues are fully protonated and positively charged.

In studying a particular biomolecule (a protein, nucleic acid, carbohydrate, or lipid) in the laboratory, the biochemist first needs to separate it from other biomolecules in the sample—that is, to purify it. Specific purification techniques are described later in the text. However, by looking at the monomeric subunits of a biomolecule, you should have some ideas about the characteristics of the molecule that would allow you to separate it from other molecules. For example, how would you separate (a) amino acids from fatty acids and (b) nucleotides from glucose?

(a) Amino acids and fatty acids have carboxyl groups, whereas only the amino acids have amino groups. Thus, you could use a technique that separates molecules on the basis of the properties (charge or binding affinity) of amino groups. Fatty acids have long hydrocarbon chains and therefore are less soluble in water than amino acids. And finally, the sizes and shapes of these two types of molecules are quite different. Any one or more of these properties may provide ways to separate the two types of compounds. (b) A nucleotide molecule has three components: a nitrogenous organic base, a five-carbon sugar, and phosphate. Glucose is a six-carbon sugar; it is smaller than a nucleotide. The size difference could be used to separate the molecules. Alternatively, you could use the nitrogenous bases and/or the phosphate groups characteristic of the nucleotides to separate them (based on differences in solubility, charge) from glucose.

Which is the conjugate base in each of the pairs below? (a) RCOOH, RCOO- (b) RNH2, RNH3+ (c) H2PO4- , H3PO4 (d) H2CO3, HCO3 -

(a) RCOO- (b) RNH2 (c) H2PO4- (d) HCO3 -

What is the net charge of Asp at pH=1?

Under standard conditions, the concentration of solutes is what?

The side chain of every ______________ amino acid residue will be found on the same face of the alpha helix

3.6th

What is the pI of glycine?

What is the pI of Lys-Glu-Ser?

6.35

Given this peptide: SEPIMAPVEYPK A) Net charge at pH 7 B) Net charge at pH 12

A) -1 B) -4

Which atoms in the alpha helix are involved in hydrogen bonding?

ALL amide protons and carbonyl oxygens

In ion-exchange chromatography with an anion exchange matrix, what proteins will elute first, and what proteins will elute last?

An anion exchange matrix is positively charged to catch negatively charged proteins. Proteins with a high pI will elute first and proteins with a low pI will elute last

Which residues would you MOST expect to find on the exterior of a water soluble protein? A) Leu, Val, Gly B) Arg, Asp, Glu C) His, Ser, Thr D) Gly, Tyr, Pro E) Ile, Lys, Val

B) Arg, Asp, Glu

Tryptophan A) negatively charged polar AA B) nonpolar aromatic AA C) positively charged polar AA D) nonpolar AA E) polar AA F) nonpolar aliphatic AA

B) nonpolar aromatic AA

Why does the mutant BPTI, which has one of its disulfide bonds cleaved, denature at a lower temperature?

Because the disulfide bond is a strong covalent bond, it requires quite a bit of energy in order to break it, but if it is already broken, less heat energy needs to be put in to unfold the protein, and so it denatures at a lower temperature

1 letter code for Cysteine

Given the following peptide (1-letter codes, N-terminus on left and C-terminus on right), what is the pI of the peptide? MESRHFYNADCAS A) 7.1 B) 4.8 C) 5.1 D) 3.9 E) none of the above

C) 5.1

3 letter code for Cysteine

Cys

1 letter code for Aspartic Acid

Proline A) negatively charged polar AA B) nonpolar aromatic AA C) positively charged polar AA D) nonpolar AA E) polar AA F) nonpolar aliphatic AA

D) nonpolar AA

If at pH 13 the charge of lysine R-group is neutral, what kind of amino acid R-groups could form a salt bridge with it?

None! Salt bridges require charge

Where are charged proteins found within a 3D protein, and why?

On the surface, because they can make favorable interactions with the solvent

1 letter code for Proline

Do proteins last forever?

Proteins have a certain half life and they do not stick around forever

1 letter code for Glutamine

3 letter code for Serine

Ser

What happens when pH is greater than pI?

The amino acid has a net negative charge

Describe the magnitide of ∆G of protein folding.

The ∆G is not that large, because proteins have to transition to folded and unfolded state, because the unfolded state is a precursor to being degraded and recycled. If the native state of the protein was so much lower in energy than the denatured state, we would never be able to recycle proteins

1 letter code for Valine

3 letter code for Valine

Val

What is the monomeric unit of a protein?

amino acid

How can we relate change in free energy to the equilibrium constant?

∆G˚ = -RT lnKeq

How can we describe change in free energy as a function of temperature?

∆G˚ = -RT lnKeq and ∆G=∆H-T∆S

In x-ray studies of crystalline peptides, Linus Pauling and Robert Corey found that the CON bond in the peptide link is intermediate in length (1.32 Å) between a typical C-N single bond (1.49 Å) and a C=N double bond (1.27 Å). They also found that the peptide bond is planar (all four atoms attached to the C—N group are located in the same plane) and that the two a-carbon atoms attached to the C-N are always trans to each other (on opposite sides of the peptide bond). (a) What does the length of the C-N bond in the peptide linkage indicate about its strength and its bond order (i.e., whether it is single, double, or triple)? (b) What do the observations of Pauling and Corey tell us about the ease of rotation about the C-N peptide bond?

(a) The higher the bond order (double or triple vs. single), the shorter and stronger are the bonds. Thus, bond length is an indication of bond order. For example, the C=N bond is shorter (1.27 Å) and has a higher order (n 2.0) than a typical C-N bond (length 1.49 Å, n 1.0). The length of the C-N bond of the peptide link (1.32 Å) indicates that it is intermediate in strength and bond order between a single and double bond. (b) Rotation about a double bond is generally impossible at physiological temperatures, and the steric relationship of the groups attached to the two atoms involved in the double bond is spatially "fixed." Since the peptide bond has considerable double-bond character, there is essentially no rotation, and the -C=O and -NH groups are fixed in the trans configuration.

What is the net charge on Asp at pH=2.1?

+0.5 (this is the pK1 value, and at this value the alpha carboxylic acid is 50% protonated and 50% deprotonated)

What is the net charge of Lys at pH 4?

What is the net charge of Lys below pK1?

What is the net charge of Asp at pH=13?

-2

What percent catalytic activity does a denatured protein have?

Bovine pancreatic trypsin inhibitor (BPTI) contains six cysteine residues that form three disulfide bonds in the native structure of BPTI. Suppose BPTI is reduced and unfolded in urea. If the reduced unfolded protein were oxidized prior to the removal of the urea, what fraction of the resulting mixture would you expect to possess native disulfide bonds?

0.0667 (5 x 3 x 1 = 15 and 1/15=0.0667)

For a weak acid with a pKa of 6.0, calculate the ratio of conjugate base to acid at a pH of 5.0.

0.1

Which of the following aqueous solutions has the lowest pH: 0.1 M HCl; 0.1 M acetic acid (pKa 4.86); 0.1 M formic acid (pKa 3.75)?

0.1 M HCl solution has the lowest pH because HCl is a strong acid and dissociates completely to H + and Cl-, yielding the highest [H].

What is the net charge of Lys at pH 9?

0.5

In a titration, how many molar equivalents of base does it take to ionize each ionizable group?

What was the rate constant that Cyrus Levinthal used for the rearrangement of a single bond?

1 x 10-13/s

List and describe 5 forces that stabilize protein 3-D structure.

1) Hydrogen bonding 2) Ionic interactions 3) Hydrophobic effect/VDW interactions 4) charged ions 5) Disulfide bonds

What are the 3 ways we can unfold proteins?

1) heating 2) change the pH 3) Add urea

A 60-residue protein and we have 20 naturally occurring amino acids. What is the total possible number of combinations of primary sequence for this protein?

1.7 x 10^78 (# choices)^(# positions)

Determine the ratio of the PROTONATED to DEPROTONATED species of Asp's R-group at pH 5.5 given the pKR of Asp = 3.86.

1/43.65

Each alpha helix consists of how many amino acids in length?

10-12, no more

When a protein is folded in its native state, what percent of catalytic activity does it have?

100%

What is the average amino acid residue weight in a protein of typical composition?

110.76 daltons

How can R groups affect alpha helices?

1R groups can de-stabilize the hydrogen bonding pattern within a helix since they are very close within 3D space. R groups with side chains that are oppositely charged can stabilize the helix

Glycine and proline are both non-polar amino acids, and both are very likely to be found at the surface of proteins. What physical characteristic of each is responsible for this observation? 1) Both glycine and proline are small. 2) Glycine is small, and proline is rigid. 3) The side chains of both these amino acids make favorable interactions with molecules near the surface of proteins. 4) Both amino acids make strong interactions with each other and therefore are likely to be found next to each other in a protein.

2 (the rigid ring of proline is well suited for turns)

What is the pI of aspartic acid?

How many inflection points will a titration curve of lysine have?

3 since it has 3 ionizable functions

How many disulfide bonds does RNase A have?

What is the pH of a solution that contains 0.20 M sodium acetate and 0.60 M acetic acid (pKa 4.76)?

4.3

Hair grows at a rate of 15 to 20 cm/yr. All this growth is concentrated at the base of the hair fiber, where a-keratin filaments are synthesized inside living epidermal cells and assembled into ropelike structures. The fundamental structural element of a-keratin is the a helix, which has 3.6 amino acid residues per turn and a rise of 5.4 Å per turn. Assuming that the biosynthesis of a-helical keratin chains is the rate-limiting factor in the growth of hair, calculate the rate at which peptide bonds of a-keratin chains must be synthesized (peptide bonds per second) to account for the observed yearly growth of hair.

42 peptide bonds per second

Calculate the approximate pI of the polypeptide N'-ATLDAK-C' (assume pK1 = 2; pK2 = 9)

6.43

Histidine is an amino acid with three titratable groups: an −NH3+ group (pKa=9.2), a −COOH group (pKa=1.8) and an imidazole (amine-like) group (pKa=6.0). What is the pI of histidine?

7.6

How many cysteine residues does RNase A have?

What is the pI of N - MCKLRVHDIY - C?

8.75

What is the pI of lysine?

9.5

Calculate the approximate pI of the polypeptide N'-MKAGHL-C'

9.75

1 letter code for Alanine

Which of the following amino acid changes would you expect to disrupt stability of a protein the least: V replaced by A or F

In an alpha helix with the following peptide sequences, which amino acid do A and L hydrogen bond to? A L D K Q N V L Y

A bonds with Q L bonds with N

What is the H-bonding pattern in the alpha helix?

A carbonyl oxygen is hydrogen bonded to the amide proton 4 residues away in the direction of the C-terminus

What is a molten globule?

A compact, partially folded intermediate that has more hydrophobic surface exposed than native state

Each point on a Ramachandran plot represents what?

A conceivable amino acid backbone configuration

How does Tm relate to protein stability?

A higher Tm means a more stable protein because it requires more thermal energy to denature

Many compounds taste sweet to humans. Sweet taste results when a molecule binds to the sweet receptor. The stronger the binding, the lower the concentration required to saturate the receptor and the sweeter a given concentration of that substance tastes. The standard free-energy change of the binding reaction between a sweet molecule and a sweet receptor can be measured in kilojoules. Sweet taste can be quantified in units of "molar relative sweetness" (MRS), a measure that compares the sweetness of a substance to the sweetness of sucrose. What is the relationship between MRS and the ∆G of the binding reaction? Would a more negative ∆G correspond to a higher or lower MRS?

A more negative G° corresponds to a larger Keq for the binding reaction, so the equilibrium Is shifted more toward products and tighter binding—and thus greater sweetness and higher MRS.

What are the differences and similarities between parallel and antiparallel beta sheets?

A parallel beta sheet is a beta sheet in which both beta strands are oriented in the same direction, with the N and C-termini of each strand going on the same direction, and an antiparallel beta sheet is one in which the two beta strands have their N-terminus and C-terminus oriented in opposite directions. In both parallel and antiparallel beta sheets, the atoms of the main chains of different beta strands form hydrogen bonds between each other, and the side chains from each beta strand are located on opposite sides of the sheet

What is a Ramachandran plot?

A plot that constitutes a map of all possible backbone conformations for an amino acid in a polypeptide. The axes of the plot consist of the two amino acid backbone bonds that are free to rotate

Mixtures of amino acids can be analyzed by first separating the mixture into its components through ion-exchange chromatography. Amino acids placed on a cation-exchange resin containing sulfonate (SO3-) groups flow down the column at different rates because of two factors that influence their movement: ionic attraction between the sulfonate residues on the column and positively charged functional groups on the amino acids, and hydrophobic interactions between amino acid side chains and the strongly hydrophobic backbone of the polystyrene resin. For each pair of amino acids listed, determine which will be eluted first from an ion-exchange column by a pH 7.0 buffer, and what the basis for separation is (a) Asp and Lys (b) Arg and Met (c) Glu and Val (d) Gly and Leu (e) Ser and Ala

A) Asp then Lys because at pH of 7, Asp has a negative charge and Lys has a positive charge B) Met, then Arg since at pH=7 Met has no net charge and Arg has a positive charge C) Glu then Val since at pH=7 Glu has -1 and Val is 0 charge D) Gly then Leu on the basis of polarity, sine leucine is more hydrophobic E) Serine, then Alanine since neither of them have a net charge, but alanine is nonpolar and will interact with the nonplar matrix (Proteins that are negatively charged will elute early due to the repulsive electrostatic interactions with the matrix, and less polar proteins pass through more slowly because of their stronger hydrophobic interactions with polystyrene)

The peptide bond... A) Has 2 resonance structures B) is shorter than a regular C-N single bond but longer than a regular C=N double bond C) a carbonyl oxygen that is a good H-bond donor D) is an amide bond E) absorbs UV light at 280 nm

A) Has 2 resonance structures B) is shorter than a regular C-N single bond but longer than a regular C=N double bond D) is an amide bond

Which of the following is CORRECT when considering the tertiary structure of globular proteins? A) Hydrophobic residues are normally on the inside and hydrophilic residues are on the outside. B) The amino acid proline never occurs in a region where the polypeptide chain bends or turns. C) All parts of the proteins can be classified as helix, β sheet or turns. D) β sheets cannot be twisted or wrapped into barrel structures. E) None of the above.

A) Hydrophobic residues are normally on the inside and hydrophilic residues are on the outside.

Which of the following statements concerning the process of spontaneous folding of proteins is false? A) It may be an essentially random process. B) It may be defective in some human diseases. C) It may involve a gradually decreasing range of conformational species. D) It may involve initial formation of a highly compact state. E) It may involve initial formation of local secondary structure.

A) It may be an essentially random process.

pI Size Binds to DNA? Protein A 7.4 82,000 Yes Protein B 3.8 21,500 Yes Protein C 7.9 23,000 No Protein X 7.8 22,000 Yes A biochemist is attempting to separate a DNA-binding protein (protein X) from other proteins in a solution. Only three other proteins (A, B, and C) are present. The proteins have the following properties: (a) protein X from protein A? (b) protein X from protein B? (c) protein X from protein C?

A) Size exclusion B) Ion-exchange C) Affinity

In a beta sheet: A) The R-groups of adjacent residues alternate by 180˚ in 3-D space perpendicular to the amide planes B) The R-groups provide more stability in the beta sheet when compared to the contributions of R-groups in an alpha helix C) Can only exist between distant (>100 residues) regions of a polypeptide as determined by the primary sequence D) The R-groups are in the same plane as the carbonyl C=O and amide N-H of the peptide bonds E) One face of the sheet is usually polar while the opposite face is non-polar

A) The R-groups of adjacent residues alternate by 180˚ in 3-D space perpendicular to the amide planes

Melanotropin has the following sequence: Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val (assume pK1=3.8 and pK2=7.9 A) Describe what a titration curve would look like B) What is the charge on it at pH 11? C) What is the charge on it at pH 5? D) What is the charge on it at pH 1? E) What is the pI?

A) The curve will have 2 inflections corresponding to 2 groups titrating near 4, one group near 7, two near 9, one near 10, and one near 12 B) -1.827 C) 2.104 D) 3.998 E) 8.813

The most important contribution to the stability of a protein's conformation appears to be the: A) entropy increase from the decrease in ordered water molecules forming a solvent shell around it B) maximum entropy increase from the ionic interactions between the ionized amino acids in the protein. C) sum of the free energies of formation of may weak interactions among the hundreds of amino acids in a protein D) sum of the free energies of formation of many weak interactions between the non- polar residues in the interior of the folded protein E) stabilizing effect of H-bonding between the carbonyl group of one peptide bond and the amino group of another

A) entropy increase from the decrease in ordered water molecules forming a solvent shell around it

Glutamic acid can be converted to to gamma-carboxyglutamate (C6H9O6) by adding a second carboxylic acid function to the last methylene (-CH2-) group in the side chain. When titrated to pH 7, what will be the effect on the overall charge on gamma-carboxyglutamate A) gamma-carboxyglutamate will be more negative compared to Glu B) gamma-carboxyglutamate more positive compared to Glu. C) There will be no difference in charge between gamma-carboxyglutamate and Glu. D) There is not enough information to answer the question. E) The answer depends on the salt concentration.

A) gamma-carboxyglutamate will be more negative compared to Glu

Aspartic acid A) negatively charged polar AA B) nonpolar aromatic AA C) positively charged polar AA D) nonpolar AA E) polar AA F) nonpolar aliphatic AA

A) negatively charged polar AA

Why is it significant that hydrogen bonds can only form at very close distances?

AAs have to be very close to each other in order to form a hydrogen bond, and they have to be in the exact correct place. If there is a mutation, such that an amino acid is not in the exact correct spot in order for a hydrogen bond to form, the bond will not form and the protein will not be as stable

What do chaperone proteins require in order to push a misfolded or almost misfolded protein back on the right path?

ATP or GTP

What type of chromatography is often used to purify DNA binding proteins and transcription factors?

Affinity (we can attach DNA to the beads)

3 letter code for Alanine

Ala

Which of the following statements about protein quaternary structure are correct? 1) It involves a complex of two or more proteins interacting with each other. 2) The subunits of the structure can be either identical or different. 3) The interactions between subunits can give rise to indefinite growth of polymeric complexes. 4) Most assemblies of protein subunits have one or more defined axis of rotation.

All

Describe the phi and psi values in the alpha helix

All of the phi values will be the same and all of the psi values will be the same, but the two values might not be completely identical

What happens when pH is less than pI?

Amino acid has a net positive charge

How are the amino acids classified?

Amino acids are classified based on the dominant chemical characteristics of their side chains, whether it be alipathic, which is where carbon groups in the side chain form straight chains, aromatic, polar side chains, basic (+ charged) side chains, and acidic (- charged) side chains

What are the acid/base characteristics of the 20 amino acids used to determine charge at a given pH?

Amino acids consist of at least a basic -NH3+ group and an acidic -COOH group, and they might have an -R group with an ionizable hydrogen atom that will also determine their charge at a given pH

What are chaotropic salts?

Ammonium sulfate, (NH4)2SO4, that does not directly interact with the protein itself, but that interact with water molecules that solvate the surface of the protein. The dissolved salt competes with the proteins for scarce water molecules, increasing the surface tension of water and therefore causing the protein to fold tighter. The reduction in protein surface area means less protein-water interactions which allows for more hydrophobic interactions between protein molecules, causing aggregation and subsequently precipitation.

What is a plasmid (expression vector) and how does it replicate in bacterial cells?

An expression vector is a modified plasmid that has a copy of the gene encoding the protein that the scientist wants to express. It also has a selectable marker gene, so cells that successfully take up the plasmid and express the protein will be resistant to a toxin in the growth medium that selects for cells that have not taken up the vector. It replicates separately from the bacterial chromosome.

3 letter code for Arginine

Arg

Which of the following amino acid changes would you expect to disrupt stability of a protein the least: Lys replaced by Asp or Arg

Arg

What makes a protein be at its lowest energy state?

As proteins are folding, they are making all different types of interactions, and what makes it the lowest energy state is maximizing the small interactions

What happens when proteins reach the end of their life?

As they are reaching the end of their life, the equilibrium starts to shift towards the unfolded state, and there are molecules that put ubiqutination tags on proteins so that proteases hydrolyze peptide bonds, destroy the proteins and recycle the amino acids

3 letter code for Asparagine

Asn

3 letter code for Aspartic acid

Asp

What is the approximate molecular weight of a protein with 682 amino acid residues in a single polypeptide chain?

Assuming that the average Mr per residue is 110 (corrected for loss of water in formation of the peptide bond), a protein containing 682 residues has an Mr of approximately 682 110 75,000.

When a protein has its maximum conformational entropy, describe the entropy of the water that it is in.

At its maximum conformational entropy, water molecules have very low entropy, because they are having to become more ordered to interact with the hydrophobic residues

Why would this alpha helix be very unstable: R L D K R R V L R A L D R Q R V R Y A L R K Q R R

At physiological pH, Arginine is + charged, and since every 3.6th R group is on the same face of the helix, the positively charged R groups of arginine will repel each other

Where are proteins the least soluble?

At their pI since they will make more interactions with themselves and be more likely to precipitate since they have both + and - charges. Above their pI they are very soluble since they are almost entirely - charged and will repel each other

Suppose the thermodynamic parameters for the folding of a protein at 1 ATM and 298K are ΔG˚ = - 10 kJ/mol; ΔS˚ = 0.05 kJ/K*mol. The equilibrium for the reaction can be expressed as U (unfolded) <--> N (native). What is ΔH˚ for this process? Does the equilibrium favor the folded or unfolded state? Would this process become more or less spontaneous as the temperature increase from 298K to 310K? A) - 4.9 kJ/mol, folded, less B) + 4.9 kJ/mol, folded, more C) - 24.9 kJ/mol, folded, more D) + 24.9 kJ/mol, unfolded, less E) Enthalpy is not correct in the above answers, unfolded, less

B) + 4.9 kJ/mol, folded, more

Citric acid is a triprotic acid with three carboxylic acid groups having pKa values of 3.1, 4.8, and 6.4. If a solution of citric acid has a pH of 5.5, what can be said about the predominant protonation state of the citric acid? (assume no partial charges, only +/- whole number integers) A) 1 carboxylic acid group is deprotonated, 2 are protonated B) 2 carboxylic acid groups are deprotonated, 1 is protonated C) all 3 carboxylic acid groups are deprotonated D) all 3 carboxylic acid groups are protonated E) the protonation state cannot be determined

B) 2 carboxylic acid groups are deprotonated, 1 is protonated

Cells contain a triprotic weak acid that accumulates as a metabolite. The pKa values for this triprotic weak acid metabolite are 2.14, 6.86 and 12.4. The metabolite has 3 different ionization states: metabolite^-2, metabolite^-1, and metabolite^0. What is the ratio of metabolite^-2/ metabolite^-1 species in a typical muscle cell where the pH is 7.2? A) 0.46 B) 2.2 C) 6.3 x 10^-6 D) 1.1 x 10^5 E) none of the above

B) 2.2 C) 6.3 x 10^-6

Which of the following statements is NOT true? A) A cofactor usually refers to a metal ion required for activity by a protein. B) A protein stripped of its cofactor or metal ion is known as a holoprotein. C) A prosthetic group is usually a small organic molecule required for activity by a protein. D) An intact protein, complete with its cofactor or metal ion, is known as a holoprotein.

B) A protein stripped of its cofactor or metal ion is known as a holoprotein.

Which statement correctly describes amphipathic (or amphiphilic) helices and sheets? A) Sheets are typically hydrophilic on one surface, whereas helices usually have both hydrophilic and hydrophobic residues evenly distributed throughout their structure. B) Amphipathic helices and sheets have predominantly hydrophilic (or hydrophobic) residues on one face. C) Helices are typically hydrophilic on one surface, whereas sheets usually have both hydrophilic and hydrophobic residues evenly distributed throughout their structure. D) Amphipathic helices and sheets have hydrophilic and hydrophobic residues evenly distributed throughout their structure.

B) Amphipathic helices and sheets have predominantly hydrophilic (or hydrophobic) residues on one face.

Which polypeptide will most likely have a pI of 9-10? A) Leu-Ala B) Arg-His C) Glu-Asp

B) Arg-His

Which polypeptide will most likely have a pI ~6 (rounding to one sig. fig.)? Mark all that are correct A) MVAHIE B) MVALNT C) MVASQI D) MVAYKT

B) MVALNT C) MVASQI

Certain amino acids destabilize or prevent formation of alpha-helices. Which AA is most likely to be found in these structures based on its charge and R-group size at pH 7. Mark all that are correct. A) Ser B) Pro C) a sequence of several glutamic acids D) a sequence of several arginines E) Ala

B) Pro C) a sequence of several glutamic acids D) a sequence of several arginines

Given the sequence of an alpha helix below, which residues would you most likely find on the same face of the helix as the N-terminal Leu? Leu-Glu-Glu-Val-Phe-Ser-Gln-Leu-Cys-Thr-His-Val-Glu-Thr-Lys A) Glu, Phe, Gln, Leu B) Val, Gln, Thr, Lys C) Phe, Ser, Gln, Leu D) Glu, Glu, Thr, Lys E) Val, Phe, Leu, Val

B) Val, Gln, Thr, Lys E) Val, Phe, Leu, Val

For amino acids with neutral R groups, at any pH below the pI of the amino acid, the population of amino acids in solution will have: A) a net negative charge. B) a net positive charge. C) no charged groups. D) no net charge. E) positive and negative charges in equal concentration.

B) a net positive charge.

Which of the following amino acid side chain is derived from another amino acid by the (1) the reduction of the R-group and (2) the addition of an amine group to R- group? A) lysine B) asparagine C) arginine D) histidine E) none of the above

B) asparagine

The uncommon amino acid selenocysteine has an R group with the structure —CH2—SeH (pKa ~ 5). The element Se is a group in Group 6 on the periodic table below Sulfur. In an aqueous solution, pH = 7.0, selenocysteine would: A) be a fully ionized zwitterion with no net charge. B) be fully ionized with a net negative charge. C) Have net 0 charge with the side chain protonated. D) be nonionic. E) not be optically active.

B) be fully ionized with a net negative charge.

Protein S will fold into its native conformation only when protein Q is also present in the solution. However, protein Q can fold into its native conformation without protein S. Protein Q, therefore, may function as a ____________ for protein S. A) subunit B) molecular chaperone C) folding intermediate D) tertiary folded protein with internal disulfide bonds E) secondary structural unit

B) molecular chaperone

What role does resonance play as it relates to the peptide bond in polypeptides?

Because of resonance, the peptide bond has a substantial amount of double bond character, so there is NO twisting around the peptide bond

How do amide planes and primary sequence influence the possible conformations of a fully folded protein?

Because of the partial double bond character of the peptide bond, the 6 atoms of the amide plane must be co-planar, and since the peptide bond cannot rotate, this limits the conformations of the fully folded protein. In addition, the preferred conformation of the polypeptide is one that allows maximum hydrogen bonding. Also, primary sequence can influence possible conformations of a fully folded protein as some bulky or highly charged R groups can restrict the protein from an alpha helix, so there are less possible conformations

Do you expect a Pro --> Gly mutation in a surface loop region of a globular protein to be stabilizing or destabilizing? Assume the mutation folds to a native-like conformation. Explain in terms of the preducted enthalpic and entropic effects of the protein on the ∆G for protein folding compared to the ∆G of folding for the wild-type protein

Because the amino acid is on the surface, loss of hydrophobic contacts in the protein core is not an issue and the AA is likely to be interacting with solvent to similar extents in both folded and unfolded states, this effects on ∆H are minimal. The ∆S for the solvent is likely to be small since side chain solvation is similar in both folded and unfolded states. ∆S for the protein changes the most due to the conformational flexibility of Gly compared to Pro. Gly will stabilize both the folded and unfolded states, but it stabilizes the unfolded state more due to tdramatic increase in conformational entropy; thus this mutation is destabilizing

Why is the mutant BPTI, which has one of its disulfide bonds cleaved, less stable as a function of temperature compared to the wild type?

Why can we not go from a fried egg to a non-fried egg?

Because the primary structure is screwed up and the albumin has formed very stable amorphous aggregates. Once the primary structure is no longer intact, it can no longer dictate any other higher order structures

Why, when Anfinsen removed BME and urea from denatured RNase A, did he get almost 100% catalytic activity?

Because the primary structure remained intact when it was unfolded, and the reason that this was not random is because the primary sequence dictates all higher order structures that form, and the reason that the correct cysteines find each other is based on where they are in the linear arrangement of their primary sequence

Which of the following statements regarding the folding of proteins is NOT true? A) Salt bridges stabilize the fold. B) Internal H bonds stabilize the fold. C) Hydrophobic residues pack together because the side chains are attracted to each other through weak Van der Waals interactions. D) Van der Waals interactions have a stabilizing, cumulative effect.

C) Hydrophobic residues pack together because the side chains are attracted to each other through weak Van der Waals interactions (they are not driven together because of the weak interactions, they are driven together to increase the entropy of the solvent)

Given the structure of a glucose molecule, which of the following explains the hydrogen bonding between glucose and water? A) H-bonds will form with glucose always being the H-bond donor B) H-bonds will form with water always being the H-bond donor C) each glucose molecule could H-bond with 17 water molecules D) each glucose molecule could H-bond with 12 water molecules E) none of the above

C) each glucose molecule could H-bond with 17 water molecules

Histidine A) negatively charged polar AA B) nonpolar aromatic AA C) positively charged polar AA D) nonpolar AA E) polar AA F) nonpolar aliphatic AA

C) positively charged polar AA

At pH 7.0, in what order would the following three peptides be eluted from a column filled with a cation-exchange polymer? Their amino acid compositions are: Protein A: Ala 10%, Glu 5%, Ser 5%, Leu 10%, Arg 10%, His 5%, Ile 10%, Phe 5%, Tyr 5%, Lys 10%, Gly 10%, Pro 5%, and Trp 10%. Protein B: Ala 5%, Val 5%, Gly 10%, Asp 5%, Leu 5%, Arg 5%, Ile 5%, Phe 5%, Tyr 5%, Lys 5%, Trp 5%, Ser 5%, Thr 5%, Glu 5%, Asn 5%, Pro 10%, Met 5%, and Cys 5%. Protein C: Ala 10%, Glu 10%, Gly 5%, Leu 5%, Asp 10%, Arg 5%, Met 5%, Cys 5%, Tyr 5%, Phe 5%, His 5%, Val 5%, Pro 5%, Thr 5%, Ser 5%, Asn 5%, and Gln 5%.

C, B, A (Protein C has a net negative charge because there are more Glu and Asp residues than Lys, Arg, and His residues. Protein A has a net positive charge. Protein B has no net charge at neutral pH. A cation-exchange column has a negatively charged polymer, so protein C interacts most weakly with the column and is eluted first, followed by B, then A.)

CS has a positive charge (at the pH of the separation) and binds to the negatively charged beads of the cation-exchange column, while negatively charged and neutral proteins pass through. CS is displaced from the column by raising the pH of the mobile phase and thus altering the charge on the CS molecules.

How do chaperone proteins work?

Chaperones bind to the hydrophobic region of the mis-folded or almost mis-folded proteins, and in an ATP or GTP dependent way, they push the protein back onto the right path

Given the following peptide (1-letter codes, N-terminus on left and C-terminus on right), what would be the overall charge at pH 9 (assume no partial charges, only +/- whole number integers)? MESRHFYNADCAS A) +2 B) +1 C) -1 D) -2 E) 0

D) -2

Biochemists now use folding funnels like this one from your text to describe the process of protein folding. Which of the following statements are true: A) Proteins begin the process of folding with high energy despite having maximum conformational entropy (the disorder of the polypeptide chain itself) because bulk solvent must become more ordered to solvate the unfolded state B) The more the conformational entropy decreases (the disorder of the polypeptide chain itself), the more the global (Ssurroundings-Spolypeptide chain) ΔS increases. C) Proteins in the native state have a higher energy than amorphous aggregates; however, few native state proteins will end up as amorphous aggregates because the energy barrier between the native state and the aggregate is so large. D) All of the statements are true E) Only statements (A) and (C) are true

D) All of the statements are true

Trypsin, a serine protease that is active in the small intestine (pH ~ 7), is concentrated with Asp residues (pKR 3.9) in its active site. It cleaves peptides by binding Lys and Arg residues in the active site. If a mutation occurred where trypsin was expressed in the stomach (pH ~ 2) but remained folded, how would the activity of trypsin change? A) Trypsin active, change in tertiary structure maintains Asp charge B) Trypsin active, metal cofactors maintain some charge on Asp C) Trypsin inactive due reduction of disulfide bonds D) Trypsin inactive due to protonation of Asp residues under low pH conditions E) Trypsin will be more active because of deprotonation of Asp side chains at pH ~ 2

D) Trypsin inactive due to protonation of Asp residues under low pH conditions

Which amino acid would most likely be found in the interior of a globular protein at pH 7? A) His B) Ala C) Asn D) Val E) Arg

D) Val (Between Ala and Val, the larger and more nonpolar it is, the more van der Waals interactions it will make within the interior of the protein, and the lower the energy will be. Valine can make more van der Waals interactions with other proteins because its side chain is longer)

Protein folding is a thermodynamically favorable process under physiological conditions because: A) there is an increase in entropy associated with protein folding. B) there is a decrease in entropy of the solvent by burying hydrophobic groups within the molecule. C) no intermediate stage disulphide bonds form during the folding process. D) of the large negative enthalpy change associated with many noncovalent interactions. E) all of the above.

D) of the large negative enthalpy change associated with many noncovalent interactions.

1 letter code for Glutamic acid

The maximum number of amino acid side chains that can potentially form an ionic bond (+||||||- interaction) with the side chain of Arg is _____at pH 2.1 ; _______at pH 7 and ______at pH 11.5 and ______at pH 13.5. (assume no partial charges, only +/- whole number integers) A) 0 ; 2; 3; 1 B) 1 ; 2 ; 4 ; 1 C) 2 ; 2; 3; 0 D) 0 ; 2; 3; 0 E) 0 ; 2; 4; 0

E) 0 ; 2; 4; 0

Because of the two coequal resonance structures of the peptide bond, the R-groups of adjacent amino acids are most often oriented approximately ______ degrees with respect to each other. A) 30˚ B) 60˚ C) 90˚ D) 120˚ E) 180˚

E) 180˚

A graduate student isolates a thermostable protein that does not readily unfold at room temperature. The wild type protein is 50% unfolded at 55˚ C, pH 6. The wild type protein possesses an Ala at position 198 of the primary sequence, a residue known to be in the hydrophobic core of the folded protein. She identifies a mutant version of the protein that is 50% unfolded at 42˚ C and possesses a single mutation at residue 198. Instead of an Ala at 198, the mutant possesses a Glu residue. Knowing the exact location of the mutant Glu residue, she engineers a compensatory (2nd) amino acid mutation that results in an almost complete reversion to wild type stability. The double mutant (Glu at position 198 and the newly added 2nd amino acid mutation) protein is 50% unfolded at 53˚ C. The compensatory (2nd) mutation she engineered to compensate for the Glu residue would most likely be: A) Trp B) Asn C) Asp D) Ser E) His

E) His

Which if the following is true regarding beta Sheets A) Hydrogen bonds in a parallel sheet involve R-groups adjacent to the peptide bond B) Phi and Psi angles in an antiparallel beta sheet are the same as those found in parallel beta sheets C) The R-groups of a beta sheet point in the same direction as the H-bonding pattern of the amide planes D) Beta sheets are less stable than alpha helices because they do not contain a specific repeating pattern of H-bonding E) None of the statements above are true

E) None of the statements above are true

Which two amino acids differ from each other by only one atom? A) Ser and Thr B) Leu and Lys C) Ala and Ser D) Asp and Asn E) Ser and Cys

E) Ser and Cys

Experiments on denaturation and renaturation after the reduction and reoxidation of the —S—S— bonds in the enzyme ribonuclease (RNase) have shown that: A) folding of denatured RNase into the native, active conformation, requires the input of energy in the form of heat. B) native ribonuclease does not have a unique secondary and tertiary structure. C) the completely unfolded enzyme, with all —S—S— bonds broken, is still enzymatically active. D) the enzyme, dissolved in water, is thermodynamically stable relative to the mixture of amino acids whose residues are contained in RNase. E) The primary sequence of RNase is sufficient to determine its specific secondary and tertiary structure

E) The primary sequence of RNase is sufficient to determine its specific secondary and tertiary structure

Threonine A) negatively charged polar AA B) nonpolar aromatic AA C) positively charged polar AA D) nonpolar AA E) polar AA F) nonpolar aliphatic AA

E) polar AA

The majority of the space on a Ramachandran plot consists of what?

Empty space with thermodynamically unfavorable combinations of psi and phi

Between enthalpy and entropy, which plays a larger role in driving protein folding?

Entropy, specifically the hydrophobic collapse, is the largest contributor to protein folding stability

Explain why ethanol (CH3CH2OH) is more soluble in water than is ethane (CH3CH3).

Ethanol is polar; ethane is not. The ethanol —OH group can hydrogen-bond with water.

1 letter code for Phenylalanine

Alanine A) negatively charged polar AA B) nonpolar aromatic AA C) positively charged polar AA D) nonpolar AA E) polar AA F) nonpolar aliphatic AA

F) nonpolar aliphatic AA

T/F: Proteins cannot self-assemble into a functional conformation after they have been denatured.

False

T/F: The interactions that stabilize multisubunit complexes are different to those that stabilize tertiary structure.

False

How do we know that ∆S is going to be overall positive for a protein folding process?

For every 1M of protein, water is at a concentration of 55M, so whether it is a micelle, lipid bilayer, or folded protein, the ∆S will always end up being big and positive because there is so much water compared to proteins or lipids

In a protein folding funnel, what does the vertical axis indicate?

Free energy

1 letter code for Glycine

3 letter code for Glutamine

Gln

3 letter code for Glutamic acid

Glu

3 letter code for Glycine

Gly

Why is the pK1 value of glycine different from that of acetic acid (both contain a single -COO- function attached to a single carbon)

Glycine contains an -NH3 group that can resonance stabilize the negative charge on the oxygen atom by withdrawing electron density, so its pK1 is lower because it will more readily release its hydrogen

1 letter code for Histidine

What was Anfinsen's experiment?

He took RNase A, and added BME and urea to completely denature the protein. Then, he removed both urea and BME to see what percent of catalytic activity he could get back, and he got back nearly 100%

3 letter code for Histidine

His

On a titration curve, what is the half equivalence point?

Horizontal line; 50% conjugate base, 50% acid

What can SDS-PAGE tell us?

How pure our protein is and the subunit composition of the protein

Why do things dissolve in water?

Hydration shell decreases attraction between two charges or polar molecules.

1 letter code for Isoleucine

What happens to an egg when we heat it up?

If we add heat, we denature the protein and the weak forces that hold the protein together, and it unfolds. In addition to it unfolding, heat breaks peptide bonds, messing up the primary sequence, and once the primary sequence is messed up, the protein cannot fold properly. Pieces of primary structures now expose their hydrophobic side chains, and then they start to coalesce and form aggregates because water does not want to solvate them. Energy levels of amorphous aggregates is VERY low, even lower than the native state, and then they come out of solution, which helps to further stabilize them. This is why the clear albumin becomes white after it is cooked, because of the aggregates

3 letter code for Isoleucine

Ile

What causes the amyloid plaques in Alzheimer's?

Improper folding due to being trapped in an intermediate state at a deep local minimum can cause aggregation because hydrophobic residues have not fully collapsed into the interior of the protein, and they will aggregate because they are exposed to solvent and this will cause the fibrils that come out of solution, and this damages the neurons, which is what causes the diseases associated with protein aggregates

How is the H-bonding pattern in the alpha helix similar to the H-bonding pattern in the beta-sheet?

In both the alpha-helix and beta-sheet, the hydrogen bond donor is the nitrogen of the amino group, and the hydrogen bond acceptor is the oxygen of the carbonyl

How many residues/turn are in an alpha helix?

In one alpha helix, it repeats after 18 residues and 5 turn, meaning that it has 3.6 residues per turn. A residue is a specific monomer in the nucleotide polymer

What happens when pH < pKa?

Ionizing groups retain their protons

Is denaturing by heat reversible or irreversible?

Irreversible, since it tends to break peptide bonds, and when peptide bonds are broken the primary structure is broken, and if the primary structure is broken, it cannot dictate any higher order folding

What questions did Cyrus Levinthal try to answer?

Is protein folding completely random? Does a protein try out every allowed combination of phi and psi and rearrange all of its bonds to try and find the correct folded 3D state?

Why is recombinant protein expression so important to modern biochemists? What does it allow them to do?

It allows them to produce proteins that they want to research in much higher concentrations than are typically found in cells, and it also allows them to create mutants if they want.

What does primary structure of a protein dictate?

It determines all higher order structures; it tells a molecule what 3D structure to assume, where and how secondary structures form, and also if and how to form quaternary structures

Why is an alpha helix no longer than 12 amino acids in length?

It eventually becomes thermodynamically unfavorable to make a longer alpha helix, because of steric hindrance, solvation, and R groups that are destabilizing to the helix. It is also hard to line up the amide planes and line up 10-12 individual dipoles. It is easier to line up just 10-12, but the more we have the more difficult it is to stabilize

Why can tyrosine be considered a polar, uncharged amino acid, as well as an aromatic amino acid

It has a bulky ring structure that makes it nonpolar, but the -OH group is ionizable under certain conditions if the pH is high enough

How does changing the pH unfold proteins?

It interrupts salt bridges between charged amino acids, and it will cause mutual repulsion between similarly charged groups. It changes where bonds can be made

At pK1, in what form is the alpha carboxylic acid of an amino acid in?

It is 50% protonated, and 50% deprotonated

At pK2, in what form is the alpha amino group of an amino acid in?

It is 50% protonated, and 50% deprotonated

Describe the interior of a folded globular protein.

It is nonpolar, and very tightly packed. The side chains of the nonpolar amino acids will maximize contact with each other to stabilize the protein and release as much energy as possible, and it wants to be as tightly packed as possible to exclude water molecules

What is Ion-exchange chromatography (IEC)?

It is used to separate molecules on the basis of their surface charge. The protein will form noncovalent interactions between the protein and the support

What is chemical equilibrium?

It is when products are being converted to reactants and reactants are being converted to products at the same rate

What was the significance of Anfinsen's experiment?

It proved that protein folding is not random, and that when the primary structure is intact, it dictates what secondary, tertiary, and possibly quaternary structures form

What does beta mercaptoethanol do?

It reduces a protein, and breaks disulfide bonds

Would it be unusual to find Pro within an alpha helix? Why or why not.

It would be unusual because prolines break alpha helices because of the nature of the side chain, becasue it does not have an amide hydrogen that can hydrogen bond with, and the cyclic nature of its side chain means that rotation around N-C is not possible.

Would it be unusual to find Ser within an alpha helix? Why or why not.

It would not be unusual, since it is an uncharged alcohol at physiological pH, and it is not super bulky and will not cause problems

Would it be unusual to find Ala within an alpha helix? Why or why not.

It would not be unusual. Its R group is just a small methyl group, so it will not cause problems

The pI of a peptide is determined by what?

Its ionizable groups

1 letter code for Lysine

What is the equilibrium for protein folding?

Keq=[N]/[U]

For protein folding to be a spontaneous equation, what must be true of Keq?

Keq=[N]/[U] For ∆G to be negative, [U] must be relatively large, and [N] must be relatively small given ∆G˚ = -RT lnKeq

1 letter code for Leucine

Histones are proteins found in eukaryotic cell nuclei, tightly bound to DNA, which has many phosphate groups. The pI of histones is very high, about 10.8. a) What amino acid residues must be present in relatively large numbers in histones? b) In what way do these residues contribute to the strong binding of histones to DNA?

Large numbers of positively charged (high pKa) groups in a protein give it a high pI. In histones, the positively charged R groups of Lys, Arg, and His residues interact strongly with the negatively charged phosphate groups of DNA through ionic interactions.

In SEC, will larger or smaller proteins elute first?

Larger

If protein folding was random, what percent of catalytic activity did Anfinsen expect when he removed BME and urea from the denatured RNase A?

Less than 1% because if it was random, there is a less than 1% chacne that the cysteine residues would partner up correctly and form the correct disulfide bonds between themselves

3 letter code for Leucine

Leu

What is Levinthal's paradox?

How did Anfinsen's experiment "solve" Levinthal's paradox?

Levinthal's paradox, which assumes that protein folding is a random process, was that RNase A, which has 10^50 possible conformations, folds fully in under 1 minute, and even if the molecule tried a new conformation every 10-13 seconds, the time it takes to generate a new conformation by the rotation of a covalent bond, it would still take over 10^30 years to try all possible conformations, so how does the protein "know" what its conformation is supposed to be. Anfinsen's experiment showed that a protein can self-assemble into its functional conformation and it needs no other information to guide it other than the information contained in its amino acid sequence, and under physiological conditions, the protein follows a thermodynamically downhill process, and it will optimize its interactions within itself and with the environment such that its native state has the lowest free energy possible.

What is the predominant type of IMF in CF4?

London

3 letter code for Lysine

Lys

Between Lysine and Asparagine, which would we most likely find on the surface of a protein at physiological pH?

Lysine; charged amino acids interact more with proteins than uncharged because electrostatic interactions are slightly more favorable than hydrogen bonding since it releases the more energy and therefore lowers the energy of the protein more

1 letter code for Methionine

Find a start signal, and write the amino acid sequence that is coded for. 5'...GCCAUGUUUCCGAGUUAUCCCAAAGAUAAAAAAGAG...3'

MFPSYPKDKKE

3 letter code for Methionine

Met

1 letter code for Asparagine

Will two cysteine residues in a protein always form a disulfide bond?

For a protein to fold, what must be the value of ∆G

Negatie

At which pH would you see the carboxylic acid group protonated, and the amino group deprotonated?

Never ever.

(Would Lys-Glu-Ser have the same pI as Ser-Glu-Lys?

No, but they would be very close. Ser-Glu-Lys will have a slightly higher pI since Serine's NH3 group has a slightly higher pKa than the NH3 group for Lysine

Do all denatured proteins take the same path to the native form?

No, protein folding is not linear, and there is not just one path.

Is protein folding linear?

No, there are multiple different pathways that a protein can take from its unfolded state to its native state

Will we find polypeptides with all of the amino acids all in the same plane?

No, we have sterics and charge-charge interactions that determine what bond angles of Φ and ψ are allowed

Can amorphous aggregates go back?

No...they are very stable, and the ∆G for their formation is so much lower and their energy is so much lower than the denatured state, they will not exist in equilibrium like normal proteins do. The aggregates that form allow for even more entropy of water

How can the binding of a charged ion stabilize proteins?

Noncovalent interactions between the ion and the protein form and stabilizes the protein

How does SEC differ from ion exchange and affinity chromatography?

Noncovalent interactions between the protein and support are negligible

If at pH 3 the charge of lysine R-group is +1, what kind of amino acid R-groups could form a salt bridge with it?

None are negatively charged, so none!

Which of the following statements regarding Anfinsen's denaturing experiments with ribonuclease A are valid? 1) Exposing the denatured protein to air oxidation and then dialysis to remove urea restored the protein to its original functionality. 2) Removing urea by dialysis and then allowing air oxidation of the denatured protein restored the protein to its original functionality. 3) Denaturing the protein with both urea and β-mercaptoethanol yielded an inactive protein. 4) Protein folding is determined by its primary sequence.

Only 2, 3, and 4

Organelles differ in size and therefore sediment at different rates during centrifugation. Larger organelles and pieces of cell debris sediment first, and progressively smaller cellular components can be isolated in a series of centrifugation steps at increasing speed. The contents of each fraction can be determined microscopically or by enzyme assay.

Osmolarity (as well as pH and temperature) affects the conformation and stability of proteins. To solubilize and renature the protein, the ammonium sulfate must be removed. In dialysis against a buffered solution containing no ammonium sulfate, the ammonium sulfate in the sample moves into the buffer until its concentration is equal in both solutions. By dialyzing against large volumes of buffer that are changed frequently, the concentration of ammonium sulfate in the sample can be reduced to almost zero. This procedure usually takes a long time (typically overnight). The dialysate must be buffered to keep the pH (and ionic strength) of the sample in a range that promotes the native conformation of the protein.

3 letter code for Phenylananine

Phe

3 letter code for Proline

Pro

What is the molecular basis for the observation that the overall charge on a protein becomes increasingly positive as pH drops and more negative as pH increases?

Protein molecules in aqueous solution become protonated as pH decreases, and proteins become more positively charged because carboxylic acids become less negatively charged as pH drops, whereas amines become more positively charge. Proteins become more negatively charged as pH increases, because acidic groups become more negatively charged while the basic groups become less positively charged

What is the reason that the hydrophobic collapse is the main driving force behind protein folding?

Proteins fold in an aqueous environment

You are given responsibility for purifying a protein. It is citrate synthase, located in the mitochondrial matrix. Following a protocol for the purification, you proceed through the steps below. As you work, a more experienced student questions you about the rationale for each procedure. Supply the answer. You proceed with the purification using the supernatant fraction that contains mostly intact mitochondria. Next, you osmotically lyse the mitochondria. The lysate, which is less dense than the homogenate, but still opaque, consists primarily of mitochondrial membranes and internal mitochondrial contents. To this lysate you add ammonium sulfate, a highly soluble salt, to a specific concentration. You centrifuge the solution, decant the supernatant, and discard the pellet. To the supernatant, which is clearer than the lysate, you add more ammonium sulfate. Once again, you centrifuge the sample, but this time you save the pellet because it contains the citrate synthase. What is the rationale for the two-step addition of the salt?

Proteins have characteristic solubilities at different salt concentrations, depending on the functional groups in the protein. In a concentration of ammonium sulfate just below the precipitation point of CS, some unwanted proteins can be precipitated (salted out). The ammonium sulfate concentration is then increased so that CS is salted out. It can then be recovered by centrifugation.

Why does the primary sequence fo a protein determine all higher order structures?

Proteins take on structure in stages defined by interactions of amino acids with each other, and the interactions that they can make are given by the amino acid sequence

Why do amorphous aggregates form?

Proteins that are incorrectly folded, leaving hydrophobic residues exposed, will tend to aggregate to cover up their hydrophobic sites. Energy levels of amorphous aggregates is VERY low, even lower than the native state, and they come out of solution, which helps keep them so stable

1 letter code for Arginine

What role do R groups play in the stabilization of the alpha helix?

R groups do not play a major role in the stabilization of secondary structures, since they do not get involved and interact until the formation of the tertiary structure, but they can destabilize any secondary structure if it is unfavorable for them to be close together in 3D space

A ________ plot describes which structures in a polypeptide are sterically possible and which are not based on the angles of rotation about the backbone Namide-Cα and Cα-Ccarbonyl bonds.

Ramachandran

Is precipitation with chaotropic salts reversible or irreversible?

Reversible

1 letter code for Serine

What is the chemical importance of SDS in visualizing proteins by SDS-PAGE?

SDS coats the proteins with a uniform negative charge so that none of them are "more negative" than others and travel based on charge instead of based on molecular weight, and it unfolds proteins by breaking noncovalent interactions so that they do not travel based on shape because they all have the same shape...linear

How do chemical reactions denature?

Salt bridges, disulfide bons, and hydrogen bonds are eliminated

What are the most important features of a protein expression vector?

Selection marker gene: a gene that encodes a protein that makes the cell that expresses it resistant to a toxin in the growth medium that selects kills cells that have not taken up the vector. Origin of replication: it is where replication starts and it determines how many copies of the vector will be made in the cell gene for protein of interest: the gene encoding the protein that they wish to have the cell produce

You are given responsibility for purifying a protein. It is citrate synthase, located in the mitochondrial matrix. Following a protocol for the purification, you proceed through the steps below. As you work, a more experienced student questions you about the rationale for each procedure. Supply the answer. You run a small sample of your fraction, now very reduced in volume and quite clear (though tinged pink), on an isoelectric focusing gel. When stained, the gel shows three sharp bands. According to the protocol, the citrate synthase is the protein with a pI of 5.6, but you decide to do one more assay of the protein's purity. You cut out the pI 5.6 band and subject it to SDS polyacrylamide gel electrophoresis. The protein resolves as a single band. Why were you unconvinced of the purity of the "single" protein band on your isoelectric focusing gel? What did the results of the SDS gel tell you? Why is it important to do the SDS gel electrophoresis after the isoelectric focusing?

Several different proteins, all with the same pI, could be focused in the "single" band. SDS polyacrylamide gel electrophoresis separates on the basis of mass and therefore would separate any polypeptides in the pI 5.6 band. SDS is a highly negatively charged detergent that binds tightly and uniformly along the length of a polypeptide. Removing SDS from a protein is difficult, and a protein with only traces of SDS no longer has its native acid-base properties, including its native pI.

You have discovered a novel protein that has a pI=5.5. To study the functional properties of this new protein, you make a mutant that contains 2 amino acid changes--a surface Phe residue on the normal protein has been replaced by His (pKa=6.1) and a surface Gln has been replaced by Glu (side chain pKa=6.0). Is the pI of the mutant protein greater, less, or the same as the pI of the normal protein?

Since the mutations replace nonionizable side chains with ionizable side chains, we can assume that any difference in charge between the wild type and mutant at pH=5.5 is due to the presence of partial charges on the mutant side chains. Using the Henderson-Hasselbach equation with the pKas, we find that at pH=5.5, the mutant protein has a charge of +.559. If a protein is + charged at a pH, the pH must be below the pI, this the pI of the mutant is higher than 5.5

Which method of chromatograpy would you use to separate an enzyme that is 54 kD from a protein that is 100 kD? Both proteins have pI values of 6.3 and are soluble in buffer at pH 7.2

Size exclusion chromatography

Why do some amino acids have 2 pKa values and others 3?

Some have 2 pKa values because they only have 2 acidic groups, but others have 3 because they have 3 acidic groups.

1 letter code for Threonine

When Cyrus Levinthal did his calculations to try to figure out how long it would take a protein if it randomly tried out every phi and psi combination, what assumption did he make?

That every bond that has free rotation can switch conformational states very quickly, and rearrange a single bond in 10^-13 seconds

You are given responsibility for purifying a protein. It is citrate synthase, located in the mitochondrial matrix. Following a protocol for the purification, you proceed through the steps below. As you work, a more experienced student questions you about the rationale for each procedure. Supply the answer. You run the dialyzed solution over a size-exclusion chromatographic column. Following the protocol, you collect the first protein fraction that exits the column and discard the fractions that elute from the column later. You detect the protein by measuring UV absorbance (at 280 nm) in the fractions. What does the instruction to collect the first fraction tell you about the protein? Why is UV absorbance at 280 nm a good way to monitor for the presence of protein in the eluted fractions?

The CS molecule is larger than the pore size of the chromatographic gel. Size-exclusion columns retard the flow of smaller molecules, which enter the pores of the column matrix material. Larger molecules flow around the matrix, taking a direct route through the column. The aromatic side chains of Tyr and Trp residues strongly absorb at 280 nm.

Why is the auto-ionization of water important?

The Kw of water is a constant, 1x10^-17. This means that since it is a constant, there will always be some H+ and OH- in solution that can be an active participant in biochemical reactions

Why does the alpha helix have an overall dipole?

The N terminal groups are all oriented going the same direction, as well as the C terminal groups, giving the N terminal side a partial + charge and the C terminal end a partial - charge. This is becasue the main chain hydrogen bonds that form orient the amide N-H and carbonyl C=O groups such that the dipoles of each of these polar bonds align

What will determine where an alpha helix finds itself in the tertiary strutrue of a protein

The R groups

In an alpha helix, how are the side chains oriented?

The R groups are pointed out and away of the helix, perpendicular to the helix

When we look down the main chain of an alpha helix, why are the R groups staggered, and not right on top of each other?

The R groups that are on the same face will be every 3.6th residue, and so becasue it is not exactly 4 residues, they will not orient themselves right on top of each other

Describe the length of a peptide bond?

The amide bond (C-N) bond is shorter than a traditional C-N bond, but a little longer than a traditional C=N bond because it is not wholly a single bond, and not wholly a double bond becasue there are 2 resonance structures that equally contribute

The axes of a Ramachandran plot consist of what?

The angles of the two backbone bonds in a polypeptide that are allowed to rotate, Φ and ψ

Which atoms are in the amide plane?

The carbonyl carbon, its oxygen, the nitrogen, and its hydrogen

What is a condensation reaction?

The elimination of a water molecule during the formation of a peptide bond and combining two amino acids by removing the -OH from the carboxylic acid of one amino acid and the -H attached to the amino group of another

You wish to purify an enzyme from bacterial cells. What percent of the total cellular protein does this enzyme represent? (1-2 sentences)

The enzyme will most likely only represent 0.01% of the total cellular protein.

Theoretical and experimental measurements show that in many cases, the contributions of ionic and hydrogen-bonding interactions to ΔH for protein folding are close to zero. Provide an explanation for this result.

The formation of intramolecular ionic of H-bonding interactions in a folded protein replace interactions between the solvent (water) and the ionic species (or H-bond donors and acceptors) in the unfolded state. The favorable ∆H obtained by the formation of intramolecular bonds in the folded protein is offset by the energy required to break many interactions with the solvent going from the unfolded to folded state

What is the hydrophobic collapse?

The hydrophobic groups pull themselves away from water and pack themselves tightly into the nonpolar core of the protein, Then, they make an array of interaction via side chains with each other. As they nucleate inward, they are making an extensive array of interactions with each other, releasing energy

What is the main driving force behind the folding of a polypeptide?

The increase in entropy of the water molecules when the protein is folded, compared to the low entropy that water has when the protein is unfolded in solution

What is Tm?

The melting temperature where 50% of molecules are denatured which can give us insight into the stability of the protein because a higher Tm means a more stable protein because it requires more thermal energy to denature

What drives the hydrophobic collapse?

The nonpolar collapse is driven by entropy because water molecules do not want to solvate the nonpolar R groups, so that is what helps tuck them into the center of the protein

What does the width of a protein folding funnel correspond to?

The number of conformational states at a given value of free energy

Which amino acids can absorb UV light at 280 nm?

The ones with a conjugated aromatic ring; tyrosine, tryptophan, and phenylalanine

How do you find the pI of an amino acid with a nonpolar R-group versus one with an acidic R-group?

The pI of an amino acid with a nonpolar R-group can be found by averaging the pKa values of the -COOH and the -NH3 groups. For an amino acid with an acidic R-group, meaning there are 3 ionizable groups, the pI can be found by averaging the pKa values that describe the ionization of the isoelectric species.

What do each of the pKa values tell you about the acid/base characteristics of an amino acid?

The pKa of the -COOH group on an amino acid has a very low value, meaning that it is acidic and readily gives up its hydrogen. The pKa of the -NH3+ group is relatively high, meaning that it does not lose their proton easily.

A claim put forth by some purveyors of health foods is that vitamins obtained from natural sources are more healthful than those obtained by chemical synthesis. For example, pure L-ascorbic acid (vitamin C) extracted from rose hips is better than pure L-ascorbic acid manufactured in a chemical plant. Are the vitamins from the two sources different? Can the body distinguish a vitamin's source?

The properties of the vitamin—like any other compound—are determined by its chemical structure. Because vitamin molecules from the two sources are structurally identical, their properties are identical, and no organism can distinguish between them. If different vitamin preparations contain different impurities, the biological effects of the mixtures may vary with the source. The ascorbic acid in such preparations, however, is identical.

The molecular weight of a protein at physiological conditions is 70kDa as determined by sedimentation equilibrium measurements and by gel filtration chromatography. The SDS-PAGE of the protein yields a single band corresponding to a molecular weight of 70 kDa. However, in the presence of reducing agent, beta metcaptoethanol, the SDS-PAGE shows 2 bands, corresponding to molecular weights of 30 kDa and 20 kDa. Describe the native protein?

The protein consists of 3 subunits with molecular weights: 30,000 Da (one subunit) and 20,000Da (2 subunits), connected with each other by disulfide bonds.

A protein has a molecular mass of 400 kDa when measured by gel filtration. When subjected to gel electrophoresis in the presence of sodium dodecyl sulfate (SDS), the protein gives three bands with molecular masses of 180, 160, and 60 kDa. When electrophoresis is carried out in the presence of SDS and dithiothreitol, three bands are again formed, this time with molecular masses of 160, 90, and 60 kDa. Determine the subunit composition of the protein.

The protein has four subunits, with molecular masses of 160, 90, 90, and 60 kDa. The two 90 kDa subunits (possibly identical) are linked by one or more disulfide bonds.

Describe ∆S as it relates to protein folding?

The protein is becoming more ordered, but as it becomes more ordered, it is releasing more and more water molecules that do not have to interact with the hydrophobic residues, and have more entropy as they are squeezed out when the protein folds

Describe protein folding.

The protein starts out in the denatured state, and goes through many intermediate states until it gets to the native state, but the protein exists in equilibrium between the unfolded, intermediate, and the native state

What is Q?

The reaction coefficient at a moment in time Q=[products]^x/[reactants]^y

A 60-residue protein has 1.7 x 10^78 possible combinations for the primary sequence of the protein. Why do we not see 1.7 x 10^78 possible combinations?

The reason that we do not find 1.7 x 10^78 primary sequences is because primary sequence determines all higher order sequences, and if certain combinations of AAs in the primary sequence make the protein less stable, evolution will select against those. For example, a polypeptide that has long strings of AAs that bear the same charge will be very unstable. For some amino acids right next to each other, there are 0 combinations of phi and psi that will stabilize

What forces stabilize the quaternary structure of a protein?

The same forces that stabilize tertiary structure: 1) disulfide bonds 2) charged ions 3) hydrophobic effect/VDW interactions 4) Ionic interactions with oppositely charged R groups attracting each other 5) hydrogen bonding between sidfe chains

How can an amide plane rotate? Is it possible?

There is no rotation around the peptide bond because it has a partial double bond character due to resonance, but there is free rotation for the entire amide plane around adjacent alpha carbons

Why do proteins have recurring secondary structures over and over?

These structures have phi and psi bond angles that are energetically favorable, and do not lead to steric clash

If both amide planes are in the plane of the board, what are the Φ and ψ values?

They are both 0˚

In a peptide bond, in what configuration are adjacent alpha carbons in?

They are in the trans configuration

What are acidic proteins?

They are rich in aspartic and glutamic acid, and have a pI below 7, so they are negatively charged at physiological pH

What are basic proteins?

They are rich in lysine, arginine, and histidine and have a pI above 7, so are positively charged at physiological pH

How do mechanical forces denature?

They eliminate weak hydrophobic interactions between amino acid side chains

How do proteins migrate (separate) in an electrophoretic field via SDS-PAGE?

They travel based on molecular weight from the negative charge to the positive charge. The smaller proteins travel further down the gel, and the larger proteins do not travel as far and are closer to the top where the proteins were initially placed

Why are certain combinations of Φ and ψ forbidden?

They will create steric clash or electrostatic repulsions

Within a folded protein molecule, how will nonpolar side chains interact?

They will interact via noncovalent van der Waals interactions, and this is thermodynamically favorable because each interaction releases a little bit of energy, but the sum of many interactions can lead to significant stabiliztion to the folded structure

3 letter code for Threonine

Thr

In protein folding, what is the ultimate goal?

To find the lowest energy state, and make as many bonds as it can, becaues the creation of each bond will release energy, and obtain the most stability

3 letter code for Tryptophan

Trp

T/F: The amino acid side chain residues in an α helix point outwards away from the center of the helix.

True

T/F: The folded conformation of proteins can be stabilized by the binding of a metal ion or cofactor.

True

3 letter code for Tyrosine

Tyr

Why does tyrosine have a 3rd pKa value, but threonine does not?

Tyrosine has an aromatic ring that threonine does not have. The resonance and electron density within the ring is drawing electron density away from the oxygen, so it can and might dissociate the hydrogen if the pH is high enough

Hydrophobic molecules do not dissolve well in water. Given that water is a very commonly used solvent, this makes certain processes very difficult: washing oily food residue off dishes, cleaning up spilled oil, keeping the oil and water phases of salad dressings well mixed, and carrying out chemical reactions that involve both hydrophobic and hydrophilic components. Surfactants are a class of amphipathic compounds that includes soaps, detergents, and emulsifiers. With the use of surfactants, hydrophobic compounds can be suspended in aqueous solution by forming micelles, which have a hydrophobic core consisting of the hydrophobic compound and the hydrophobic "tails" of the surfactant; the hydrophilic "heads" of the surfactant cover the surface of the micelle. A suspension of micelles is called an emulsion. The more hydrophilic the head group of the surfactant, the more powerful it is—that is, the greater its capacity to emulsify hydrophobic material. When you use soap to remove grease from dirty dishes, the soap forms an emulsion with the grease that is easily removed by water through interaction with the hydrophilic head of the soap molecules. Likewise, a detergent can be used to emulsify spilled oil for removal by water. And emulsifiers in commercial salad dressings keep the oil suspended evenly throughout the water-based mixture. There are some situations in which it would be very useful to have a "switchable" surfactant: a molecule that could be reversibly converted between a surfactant and a nonsurfactant. Imagine such a "switchable" surfactant existed. How would you use it to clean up and then recover the oil from an oil spill?

Use the substance in its surfactant form to emulsify the spilled oil, collect the emulsified oil, then switch to the nonsurfactant form. The oil and water will separate and the oil can be collected for further use.

1 letter code for Tryptophan

How do we elute proteins from an ion-exchange column?

We can elute by changing the pH of the solution, which changes the surface charge on proteins, weakening the interaction between proteins and the matrix. We can also increase the ionic strength of the mobile phase. Soluble ions (NaCl) compete for binding to the charged functional groups on the matrix and as the concentration of soluble ions increases, they outcompete and displace the protein from the matrix

You are given responsibility for purifying a protein. It is citrate synthase, located in the mitochondrial matrix. Following a protocol for the purification, you proceed through the steps below. As you work, a more experienced student questions you about the rationale for each procedure. Supply the answer. You pick up 20 kg of beef hearts from a nearby slaughterhouse (muscle cells are rich in mitochondria, which supply energy for muscle contraction). You transport the hearts on ice, and perform each step of the purification on ice or in a walk-in cold room. You homogenize the beef heart tissue in a high-speed blender in a medium containing 0.2 M sucrose, buffered to a pH of 7.2. Why do you use beef heart tissue, and in such large quantity? What is the purpose of keeping the tissue cold and suspending it in 0.2 M sucrose, at pH 7.2? What happens to the tissue when it is homogenized?

We use beef heart tissue, because the protein is found in the mitochondria, which are abundant in cells with high metabolic activity, such as hearts. We need a lot, becasue cells contain thousands of different proteins, and no single protein is present in high concentration, so the specific activity of CS is low. In order to purify a lot, we need a lot of tissue. The cold inhibits the lysosomal enzymes that would destroy the sample Sucrose is used in the homogenization buffer to create a medium that is isotonic with the organelles. This prevents diffusion of water into the organelles, causing them to swell, burst, and spill their contents. A pH of 7.2 helps to decrease the activity of lysosomal enzymes and maintain the native structure of proteins in the sample. Homogenization breaks open the heart muscle cells, releasing the organelles and cytosol

Why would we, or why would we not find water in the center of the alpha helix?

We would not find water in the center of the alpha helix since if water gets into the center of the helix, it disrupts the hydrogen bonding pattern, and so the atoms on the interior of the helix pack as tightly as they possible can in order to exclude water

How are R groups that are located on adjacent alpha carbons arranged about a peptide bond?

When amino acids are linked together by a peptide bond and are assembled into a polypeptide, if we look at all of the adjacent R groups hanging off of adjacent alpha carbons, 99.9% of the time they will be in the trans configuration, because if they were in the cis-configuration, they would lead to steric clash

The denaturing of proteins occurs when what happens?

When stabilizing forces are altered and the protein is unfolded

On a titration curve, what is the equivalence point?

When the moles of base are equal to the moles of acid; vertical line

Aspirin is a weak acid with a pKa=3.5. It is absorbed into the blood through the cells lining the stomach and the small intestine. Absorption requires passage through the plasma membrane, the rate of which is determined by the polarity of the molecule: charged and highly polar molecules pass slowly, whereas neutral hydrophobic ones pass rapidly. The pH of the stomach contents is about 1.5, and the pH of the contents of the small intestine is about 6. Is more aspirin absorbed into the bloodstream from the stomach or from the small intestine?

With a pKa of 3.5, aspirin is in its protonated (neutral) form at pH below 2.5. At higher pH, it becomes increasingly deprotonated (anionic). Thus, aspirin is better absorbed in the more acidic environment of the stomach.

1 letter code for Tyrosine

Will acidic and basic proteins migrate in the same manner in SDS-PAGE?

Yes! By adding SDS, they have the same negative charge

At pH 7.2, will an NH3+ group that has a pKa of 8.5 ever be nonprotonated?

Yes, the protonated and deprotonated forms of each ionizable group are in equilibrium, so a small bit of the population will contain a deprotonated NH3+.

Can one amino acid belong to more than one subcategory? Cite an example

Yes, tyrosine has an aromatic ring, and at high pH values can release its -H of its -OH group, so it can also belong in the polar, negatively charged acidic amino acid group

What does a nucleoside consist of?

a base and a sugar

What is a residue?

a monomeric unit of a polypeptide or polynucleotide chain

What is a cation exchanger?

a negatively charged matrix to catch positively charged proteins

What is a hydrogen bond?

a non-covalent interaction in which a hydrogen atoms is partially shared between 2 electronegative atoms

What is the monomeric unit of a nucleic acid?

a nucleotide

What is an anion exchanger?

a positively charged matrix to catch negatively charged proteins

What is an exothermic reaction?

a reaction in which heat is released

What is an endothermic reaction?

a reaction in which heat is taken up

What is a beta strand?

a stretch of polypeptide chain typically 3 to 10 amino acids long with backbone in an extended conformation

What is a torsion angle?

a type of dihedral angle used to define parts of a molecule joined by a chemical bond

A new amino acid similar to K has been discovered that possesses a side chain with two ionizable functions. The pKR values for these functions are pKR 1= 9.78 and pKR 2 = 11.42. Assume pK1 and pK2 are identical to K. (a) Calculate the pI of this amino acid. (b) How many molar equivalents of OH- must be added to this amino acid for it to posses a net -1 charge?

a) 10.6 b) 4

What is the charge of N'-HKLFVAG-C' at A) pH 1 B) pH 7 and C) pH 10?

a) 3 b) 1 c) 0

A peptide has the sequence Glu-His-Trp-Ser-Gly-Leu-Arg-Pro-Gly (a) What is the net charge of the molecule at pH 3, 8, and 11? (b) Estimate the pI for this peptide.

a) 3: +2, 8: 0, 11: -1 b) 7.8

What is the pH of a solution that has an H concentration of (a) 1.75 x 10^-5 mol/L (b) 6.50 x 10^-10 mol/L (c) 1.0 x 10^-4 mol/L (d) 1.50 x 10^-5 mol/L

a) 4.76 b) 9.19 c) 4 d) 4.82

Some natural proteins are rich in disulfide bonds, and their mechanical properties (tensile strength, viscosity, hardness, etc.) are correlated with the degree of disulfide bonding. (a) Glutenin, a wheat protein rich in disulfide bonds, is responsible for the cohesive and elastic character of dough made from wheat flour. Similarly, the hard, tough nature of tortoise shell is due to the extensive disulfide bonding in its a-keratin. What is the molecular basis for the correlation between disulfide-bond content and mechanical properties of the protein? (b) Most globular proteins are denatured and lose their activity when briefly heated to 65˚ C. However, globular proteins that contain multiple disulfide bonds often must be heated longer at higher temperatures to denature them. One such protein is bovine pancreatic trypsin inhibitor (BPTI), which has 58 amino acid residues in a single chain and contains three disulfide bonds. On cooling a solution of denatured BPTI, the activity of the protein is restored. What is the molecular basis for this property?

a) Disulfide bonds are covalent bonds, which are much stronger than the noncovalent interactions (hydrogen bonds, hydrophobic interactions, van der Waals interactions) that stabilize the three-dimensional structure of most proteins. Disulfide bonds serve to cross-link protein chains, increasing stiffness, hardness, and mechanical strength. (b) As the temperature is raised, the increased thermal motion of the polypeptide chains and the vibrational motions of hydrogen bonds ultimately lead to thermal denaturation (unfolding) of a protein. Cystine residues (disulfide bonds) can, depending on their location in the protein structure, prevent or restrict the movement of folded protein domains, block access of solvent water to the interior of the protein, and prevent the complete unfolding of the protein. Refolding to the native structure from a random conformation is seldom spontaneous, owing to the very large number of conformations possible. Disulfide bonds limit the number of conformations by allowing only a few minimally unfolded structures, and hence the protein returns to its native conformation more easily upon cooling.

Consider: 1Ile-2Ala-3His-4Thr-5Tyr-6Gly-7Pro-8Phe-9Hly-10Ala-11Ala-12Met-13Cys-14Lys-15Trp-16Glu-17Ala-18Gln-19Pro-20Asp-21Gly-22Met-23Glu-24Cys-25Ala-26Phe-27His-28Arg A) Where might intrachain disulfide cross-linkages be formed? B) Assuming that this sequence is part of a larger globular protein, indicate the probable location (the external surface or interior of the protein) of the following amino acid residues: Asp, Ile,Thr, Ala, Gln, Lys. Explain your reasoning.

a) Intrachain disulfide cross-linkages can form only between residues 13 and 24 (Cys residues). (c) Amino acids with ionic (charged) or strongly polar neutral groups (e.g., Asp, Gln, and Lys in this protein) are located on the external surface, where they interact optimally with solvent water. Residues with nonpolar side chains (such as Ala and Ile) are situated in the interior, where they escape the polar environment. Thr is of intermediate polarity and could be found either in the interior or on the exterior surface

Consider an alpha helix with the sequence N'-ACYMTCKTSING-C'. (a) The 3rd residue is Y, which residue is it forming a H-bond with? (b) The 5th residue is T, which residue is it forming a H-bond with?

a) K b) S (It forms a hydrogen bond with the residue 4 positions away in the direction of the C-terminus)

Using one letter codes, list ALL amino acids whose R-groups can form a salt bridge with the side chain of E at (a) pH 3 (b) pH 7 (c) pH 13

a) None b) K and R c) None

Pepsin is the name given to a mix of several digestive enzymes secreted (as larger precursor proteins) by glands that line the stomach. These glands also secrete hydrochloric acid, which dissolves the particulate matter in food, allowing pepsin to enzymatically cleave individual protein molecules. The resulting mixture of food, HCl, and digestive enzymes is known as chyme and has a pH near 1.5. A) What pI would you predict for the pepsin proteins? B) What functional groups must be present to confer this pI on pepsin? C) Which amino acids in the proteins would contribute such groups?

a) Pepsin proteins have a relatively low pI (near the pH of gastric juice) in order to remain soluble and thus functional in the stomach. As pH increases, pepsins acquire a net charge and undergo ionic interactions with oppositely charged molecules (such as dissolved salts), causing the pepsin proteins to precipitate. b) A low pI requires large numbers of negatively charged (low pKa) groups. c) These are contributed by the carboxylate groups of Asp and Glu residues.

In considering protein secondary structure which of the following is INCORRECT? a) The 310 helix is right-handed and often contains proline residues. b) An α helix repeats after 18 residues and has 3.6 residues per turn. c) The most common structures are the α helix and the β sheet. d) The β strands can be in either parallel or antiparallel configuration. e) A network of main-chain hydrogen bonds connect β strands in a β sheet.

a) The 310 helix is right-handed and often contains proline residues.

At a pH equal to the isoelectric point of alanine, the net charge on alanine is zero. Two structures can be drawn that have a net charge of zero, but the predominant form of alanine at its pI is zwitterionic. (a) Why is alanine predominantly zwitterionic rather than completely uncharged at its pI? (b) What fraction of alanine is in the completely uncharged form at its pI?

a) The pI of alanine is well above the pKa of the a-carboxyl group and well below the pKa of the a-amino group. Hence, at pH pI, both groups are present predominantly in their charged (ionized) forms. b) 1 in 2.19 x 10^7.

"The amino acid residues in protein molecules are exclusively L stereoisomers." It is not clear whether this selectivity is necessary for proper protein function or is an accident of evolution. To explore this question, Milton and colleagues published a study of an enzyme made entirely of D stereoisomers. The enzyme they chose was HIV protease, a proteolytic enzyme made by HIV that converts inactive viral pre-proteins to their active forms. Previously, Wlodawer and coworkers had reported the complete chemical synthesis of HIV protease from L-amino acids (the L-enzyme). Normal HIV protease contains two Cys residues at positions 67 and 95. Because chemical synthesis of proteins containing Cys is technically difficult, Wlodawer and colleagues substituted the synthetic amino acid L-alpha-amino-n-butyric acid (Aba) for the two Cys residues in the protein. The newly synthesized protein was denatured by dissolving it in 6 M guanidine HCl, and then allowed it to fold slowly by dialyzing away the guanidine against a neutral buffer (10% glycerol, 25 mM NaPO4, pH 7). This protein was made by substituting two Cys residues with a synthetic amino acid that does not contain sulfur. (a) There are many reasons to predict that a protein synthesized, denatured, and folded in this manner would not be active. Give three reasons. (b) Interestingly, the resulting L-protease was active. What does this finding tell you about the role of disulfide bonds in the native protein molecule? (c) In their new study, Milton and coworkers synthesized HIV protease from D-amino acids, using the same protocol as the earlier study. Formally, there are three possibilities for the folding of the D-protease: it would give (1) the same shape as the L-protease, (2) the mirror image of the L-protease, or (3) something else, possibly inactive. For each possibility, decide whether or not it is a likely outcome and defend your position.

a) There are many important differences between the synthesized protein and HIV protease produced by a human cell, any of which could result in an inactive synthetic enzyme: (1) Although Aba and Cys have similar size and hydrophobicity, Aba may not be similar enough for the protein to fold properly. (2) HIV protease may require disulfide bonds for proper functioning. (3) Many proteins synthesized by ribosomes fold as they are produced; the protein in this study folded only after the chain was complete. (4) Proteins synthesized by ribosomes may interact with the ribosomes as they fold; this is not possible for the protein in the study. (5) Cytosol is a more complex solution than the buffer used in the study; some proteins may require specific, unknown proteins for proper folding. (6) Proteins synthesized in cells often require chaperones for proper folding; these are not present in the study buffer. (7) In cells, HIV protease is synthesized as part of a larger chain that is then proteolytically processed; the protein in the study was synthesized as a single molecule. b) Because the enzyme is functional with Aba substituted for Cys, disulfide bonds do not play an important role in the structure of HIV protease. c) Model 1: it would fold like the L-protease. Argument for: the covalent structure is the same (except for chirality), so it should fold like the L-protease. Argument against: chirality is not a trivial detail; three-dimensional shape is a key feature of biological molecules. The synthetic enzyme will not fold like the L-protease. Model 2: it would fold to the mirror image of the L-protease. For: because the individual components are mirror images of those in the biological protein, it will fold in the mirror-image shape. Against: the interactions involved in protein folding are very complex, so the synthetic protein will most likely fold in another form. Model 3: it would fold to something else. For: the interactions involved in protein folding are very complex, so the synthetic protein will most likely fold in another form. Against: because the individual components are mirror images of those in the biological protein, it will fold in the mirror image shape.

The R-groups of amino acids are not involved in the formation of secondary structure. If true, then any polypeptide with any sequence should be able to form an alpha helix. In 3 sentences or fewer, explain why: (a) N'-WWWWWWWWWWWW-C' does not form a stable alpha helix. (b) why does N'-EEEEEEEEEEEEE-C' form an alpha helix at pH < 4, but not at higher pH values.

a) W is big, the side chains collide and this is sterically hindering and it will disrupt the alpha helix b) below pH 4, the -COOH group of E's side chain is protonated, and there will be no electrostatic repulsions, but above pH 4, -COO is negatively charged, and there will be unfavorable electrostatic repulsions that disrupt the alpha helix

List ALL amino acids whose R-groups can form a salt bridge with the side chain of K at (a) pH 3 (b) pH 7 (c) pH 13

a) none b) Aspartic acid (D) and glutamic acid (E) c) Nothing! Salt bridges require charge

Classify glutamic acid as acidic, basic, neutral polar, or neutral nonpolar

acidic (its R-group contains the functional group -COOH)

When does the hydrophobic collapse happen?

after secondary structures have formed

Which of the following statements about globular proteins are true? 1) The protein folds to make itself as compact as possible. 2) The packing of the protein is such that hydrophilic residues appear on the surface where they can interact with an aqueous environment. 3) Irregularities of the protein's surface allow for the formation of clefts, which are often where the protein promotes a chemical transformation. 4) Regions of secondary structures folding on one another are examples of the protein's tertiary structure.

all

When Anfinsen removed BME and urea from denatured RNase A, what percent of catalytic activity did he get?

almost 100%

What is a dihedral angle?

an angle between 2 intersecting planes

Give two reasons to explain why a proline residue in the middle of an α-helix is predicted to be destabilizing to the helical structure. a) Pro is nonpolar amino acid that destabilizes polar core of the protein. b) Pro does not have the α−NH group that acts as a stabilizing H-bond donor in the middle of the helix. c) Pro is nonpolar amino acid that does not connect turns of the α-helix. d) Pro is not able to adopt the ideal ϕ and ψ angles for an α -helix. e) Insertion of Pro gives an exceptional conformational rigidity to the protein chain chain.

b) Pro does not have the α−NH group that acts as a stabilizing H-bond donor in the middle of the helix. d) Pro is not able to adopt the ideal ϕ and ψ angles for an α -helix. (there are specific angles that are allowed for an α-helix that will minimize steric crowding and minimize the energy requirement, becasue steric crowding costs energy)

Why do proteins that are stuck in an "off-pathway" intermediate tend to aggregate?

b. If proteins are stuck in intermediate states, hydrophobic residues tend to aggregate because they are exposed to solvent, and this leads to fibrils that come out of solution and cause disease (i.e. amyloid plaques in Alzhiemers)

Classify arginine as acidic, basic, neutral polar, or neutral nonpolar

basic

Classify lysine as acidic, basic, neutral polar, or neutral nonpolar

basic

Why do all proteins in SDS-PAGE migrate from the negative to the positive pole?

because SDS binds to proteins and confers the same negative charge to mass ratio

Match the following characteristic to α helices, β sheets, or both: extensive H-bonding network

both

How is tertiary structure determined?

by the primary structure

Which short peptide(s) is (are) consistent with a titration curve with 2 inflection points? One is at pH 2.3, the second at pH 9.6. a) Ala-Glu-Val b) Ala-Tyr-Ser c) Ala-Thr-Pro d) His-Ala-Leu e) None of the above

c) Ala-Thr-Pro

On a molar basis (assume equal concentrations), which of the peptides would absorb the most UV light at 280nm? a) Asp-Gly-Phe-His-Pro b) Leu-Trp-Phe-His-Ser c) Ala-Tyr-His-Tyr-Phe

c) Ala-Tyr-His-Tyr-Phe

How does urea unfold proteins?

coats polypeptide and breaks noncovalent 3D interactions

What kind of bond is the peptide bond?

covalent bond

How is the alpha helix primarily stabilized? a) Hydrophobic packing of R groups in the interior b) R group interactions perpendicular to the axis of the helix c) Ionic interactions between functional groups d) H-bonding parallel to the axis of the alpha helix e) H-bonding between R groups

d) H-bonding parallel to the axis of the alpha helix

For an amino acid such as aspartic acid, what impact do you expect the two neighboring carboxylic acids to have on the pKa values for each? a) The acid with the higher pKa value would decrease the pKa value for the other acid. b) The neighboring acids would have no impact on each other's pKa values. c) The acid with the lower pKa value would decrease the pKa value for the other acid. d) The acid with the lower pKa value would increase the pKa value for the other acid.

d) The acid with the lower pKa value would increase the pKa value for the other acid. (the presence of one negative charge is likely to make the formation of a second negative in close proximity less favorable because of the charge-charge repulsions)

What is the only covalent force that stabilizes proteins?

disulfide bond

What is the only strong force to stabilize proteins?

disulfide bond

The side chain of ________ has a pKa in the physiological pH range and is therefore often involved in proton transfer during enzymatic catalysis.

histidine (At physiologial pH, histidine will exist in both protonated and deprotonated forms)

________ between amide protons and carbonyl oxygens is necessary to stabilize a regular folding of protein secondary structure.

hydrogen bonding

How does heating denature?

hydrogen bonding and hydrophobic interactions are eliminated, as well as peptide bonds

What are the common stabilizing forces of the two main types of secondary structure?

hydrogen bonds

What stabilizes the beta sheet?

hydrogen bonds between the amide proton and the carbonyl oxygen

Where is glycogen stored?

in the liver and skeletal muscle

How does the melting point change as the strength of intermolecular forces increase?

increase

Where does the energy to get out of an off-path intermediate on the way to the native state of a protein come from?

internal enthalpy; generally by rearrangement of non-covalent interactions because once optimal interactions are met the energy that comes out in the form of H-bonds, ionic bonds, etc. is enough to overcome local energy minima

What happens when pH > pKa?

ionizing groups lose their protons

What is a "face" of an alpha helix or beta sheet?

it is a side of the helix or sheet that has R groups of similar chemical nature and polarity

What is Keq?

it quantifies the position of a chemical equilibrium, the concentration of products to the concentration of reactants when the reaction is at equilibrium (products are converting to reactants and reactants are converting to products at the same rate) Keq=[products]/[reactants]

What does urea do?

it unfolds a protein

Would it be unusual to find Glu within an alpha helix? Why or why not.

it would be unusual, since at physiological pH, the R groups are deprotonated, and the negative charges will try to push away from each other, which will tear apart the hydrogen bonding pattern that holds together the helix

Would it be unusual to find Arg within an alpha helix? Why or why not.

it would be unusual, since at physiological pH, the R groups are positively charged, and the positive charges will try to push away from each other, which will tear apart the hydrogen bonding pattern that holds together the helix

What is a hydrogen bond acceptor?

lone pairs on nitrogens, oxygens, and possibly sulfurs that are accepting the positively charged hydrogen

Classify alanine as acidic, basic, neutral polar, or neutral nonpolar

neutral nonpolar

Classify phenylalanine as acidic, basic, neutral polar, or neutral nonpolar

neutral nonpolar

Classify proline as acidic, basic, neutral polar, or neutral nonpolar

neutral nonpolar

Classify valine as acidic, basic, neutral polar, or neutral nonpolar

neutral nonpolar

Classify methionine as acidic, basic, neutral polar, or neutral nonpolar

neutral nonpolar (long hydrocarbon chain, and sulfur has the same electronegativity as carbon)

Classify tryptophan as acidic, basic, neutral polar, or neutral nonpolar

neutral nonpolar (the presence of the bulky aromatic R group diminishes the presence of the -NH)

Classify tyrosine as acidic, basic, neutral polar, or neutral nonpolar

neutral nonpolar or neutral polar (the presence of the bulky aromatic R group diminishes the presence of the -OH)

Classify asparagine as acidic, basic, neutral polar, or neutral nonpolar

neutral polar

Classify cysteine as acidic, basic, neutral polar, or neutral nonpolar

neutral polar

Classify glutamine as acidic, basic, neutral polar, or neutral nonpolar

neutral polar

Classify serine as acidic, basic, neutral polar, or neutral nonpolar

neutral polar

Classify threonine as acidic, basic, neutral polar, or neutral nonpolar

neutral polar (presence of the electronegative oxygen atom in its R-group)

Where are polar amino acids found within a protein? And why?

on the surface because they can make favorable interactions with the solvent

In biochemical systems, what atoms are usually hydrogen bond acceptors?

oxygen, nitrogen, and rarely sulfur

In biochemical systems, what atoms are usually hydrogen bond donors?

oxygen, nitrogen, and sulfur

Acidic proteins have what type of pI?

pI < 7

Basic proteins have what type of pI?

pI > 7

Rank these hydrocarbons in order of decreasing boiling point: 2,2,4-trimethylpentane (C8H18), octane (C8H18), nonadecane (C19H40), ethane (C2H6), paraffin (C36H74)

paraffin (C36H74), nonadecane (C19H40), octane (C8H18), 2,2,4-trimethylpentane (C8H18), ethane (C2H6)

Which of the following peptides is more likely to take up an alpha-helical structure, and why? (a) LKAENDEAARAMSEA (b) CRAGGFPWDQPGTSN

peptide A has five Ala residues (most likely to take up an alpha-helical conformation), and peptide B has five Pro and Gly residues (least often found in an alpha helix).

What is affinity chromatography?

protein of interest is adsorbed onto a ligand, typically a substrate or inhibitor, and the interactions between the protein of interest and the ligand are very specific, so most contaminants will not interact with the matrix and forms noncovalent interactions between the protein and the support

What is the most abundant macromolecule within a cell?

proteins

What are chaperone proteins?

proteins that are protein folding catalysts that manage the folding of other proteins. They help rescue proteins that have gone off path during the folding process

What are the 4 types of biomolecules?

proteins, carbohydrates, nucleic acids, and lipids

What is an alpha helix?

repeating pattern of hydrogen bonding forces that stabilize the helix

Is denaturing with urea reversible or irreversible?

reversible

What is size exclusion chromatography (SEC)?

separates proteins based on apparent size (which is correlated to the length of the amino acid sequence)

What is the secondary structure of a protein?

small section of organized protein structure, such as helices

What is the hybridization of oxygen in a water molecule?

sp3

What is the matrix in SEC?

spherical beads with pores in the surface to catch proteins

Which part of an animo acid characterizes whether that amino acid is neutral, acidic, or basic, polar, or nonpolar?

the R group

In an amino acid, off of which atom does the R group hang off of?

the alpha carbon

How are R groups oriented in a beta sheet?

the are perpendicular to the hydrogen bonding pattern; there is an alternating pattern of R groups because of the nature of phi and psi angles, going into and out of the plane of the board for both parallel and antiparallel beta sheets

What happens if the Φ and ψ angles are both 0˚?

the carbonyl oxygens and amide protons sterically clashx

What is an alpha carbon?

the central carbon in an amino acid that is one position away from the carboxylic acid group, and the carbon that the R group hangs off of

What determines where an amino acid will end up within the 3D folded protein?

the chemical nature of its side chain

Which state of a protein has the highest conformational entropy?

the denatured state

Which state of a protein has the highest energy?

the denatured state

What is the native state of a protein?

the folded state

What is the quaternary structure of a protein?

the interaction between 2 separate protein strands

What state of the protein do chaperones interact with?

the intermediates

In SEC, the first proteins to elute from the column are

the largest (the volume of bigger required to elute a protein depend on what fraction of the column that the protein can occupy. The more volume of the column it can occupy, the smaller it is, and the more buffer needed to elute it, and the later it will elute. Small proteins can occupy more volume of the column since they can occupy space within beads and between beads)

What is the primary structure of a protein?

the linear sequence of amino acids in a polypeptide chain

What is the tertiary structure of a protein?

the overall fold of a single protein strand with the contributions of all of the R groups and all of the other functional groups that are capable of hydrogen bonding

What is pK1?

the pH where the alpha carboxylic acid is deprotonated/protonated

What is pK2?

the pH where the amino acid group is deprotonated/protonated

What is the denatured state of a protein?

the unfolded state

Φ and ψ angles are measured by what?

their deviation from a completely planar state

How are hydrogen bonds oriented in a beta sheet?

they are in the same plane as the beta sheet

In the alpha helix, how are the hydrogen bonds oriented with respect to the helix?

they are parallel to the axis of the helix

Why can amorphous aggregates ever "go back?"

they are very stable, and the ∆G for their formation is so much lower and their energy is so much lower than the denatured state, they will not exist in equilibrium like normal proteins do. The aggregates that form allow for even more entropy of water. In addition, when they come out of solution they can no longer be solvated

Where are nonpolar alipathic amino acids found within a protein?

they will coalesce on the interior of the protein and interact with each other

Disulfide bonds require what in order to form?

two cysteine residues in very close proximity

In a hydrogen bond, what type of atoms "share" hydrogen?

two electronegative atoms

What type of distance do hydrogen bonds act over?

very small distances

Lipid bilayers form because of what?

water

Match the following characteristic to α helices, β sheets, or both: 5.4Å/turn

α helices

Match the following characteristic to α helices, β sheets, or both: large dipole moment

α helices (the arrangement of the main-chain hydrogen bonds in the alpha helix orients the N-H and C=O groups such that the dipole moments for each of these polar bonds alive and give rise to a helical dipole moment)

Match the following characteristic to α helices, β sheets, or both: antiparallel arrangement

β sheets (duh, an alpha helix is made from one polypeptide strand)

How can we describe change in enthalpy as a function of temperature?

∆G=∆H-T∆S

How can we describe change in entropy as a function of temperature?

∆G=∆H-T∆S

The subunits of some multimeric proteins actually associate and form quaternary structure more readily as temperature increases. Show one of the equations from our "life obeys the laws of thermodynamics" discussion and explain in one sentence why higher temperature would make the association of the subunits more favorable. Assume ∆H remains approximately the same before and after subunit association.

∆G=∆H-T∆S and ∆G˚ = -RT lnKeq As the temperature increases, ∆G gets more spontaneous, and therefore more energy is released from the system to the surroundings, making it more favorable

The ∆G˚ of the reaction to split a 6-carbon sugar into 2 x 3-carbon sugars in glycolysis is +26 kJ/mol. Why then, does this reaction still take place?

∆Gcell = ∆G˚ + RT lnQ where Q = [products]/[reactants]. ∆Gcell has to take into account the concentration of products and reactants because the concentration is not 1M since inside of a cell it is not under standard conditions. If we keep products of products low, we can create a reaction that under standard conditions will never happen, under cellular conditions, by modulating metabolic flux of the solutes (products and reactants) allows the reaction to happen, because ∆Gcell ends up being negative

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