Chapter 4
Steps in Anfisen's experiment
***LOOK TEXTBOOK***
14. What are the two phenotypical consequences produced by protein-folding diseases?
1. The degradation of a misfolded protein, known as a loss of function effect because the activity of the particular protein is missing. 2. Protein aggregation, known as gain of function effect because these proteins add a process to the cell. Gain of function protein folding phenotypes can result from missense mutations or from accumulation of misfiled wild-type proteins.
All free amino acids are ___.
Acids and bases.
9. What are the three most common amino acids found in alpha helixes, beta strands, and beta turns, respectively?
Alpha helix: glutamate, alanine, and leucine. Beta strands: valine, isoleucine, and tyrosine. Beta turns: glycine, asparagine, and proline.
7 hydrophobic amino acids, lacking polar groups
Glycine (included because it's most chemically neutral), alanine, proline, valine, leucine, isoleucine, methionine.
What is the affinity of the 5 charged amino acids?
Hydophilic
Definition of oxidized
Lost electrons. Reduced state is gaining electrons.
Which amino acids contain sulfur?
Methionine, cysteine.
Amino acids with bulky aromatic side chains
Phenylalanine, tyrosine, tryptophan
11. What is thought to be the dominant mechanism of protein folding? Why can it not be random?
Preferred folding pathways. Random folding would be too time consuming; for example, a protein of 100 amino acids sampling three possible phi and psi angles for each amino acid would take billions of years to fold.
α helices, β strands, and β turns are examples of __________ structure.
Secondary
What forms amide bonds in proteins?
The internal NH2 and COOH groups
Typical pKa values of terminal residues (which are titratable groups)
α carboxyl: 3.1 α amino: 8.0
14. Answer the following questions based on these four primary protein sequences: 1. Asp-gln-leu-glu-lys-glu-leu-gln-ala-leu-glu-lys-glu-leu-ala 2. phe-gln-ile-asp-met-glu-leu-lys-val-asn-leu-asp-phe-arg-ala 3. ala-gli-tyr-gly-pro-asn-leu-phe-ala-val-ile-lys-asn-cys-ala 4. phe-asn-ser-val-leu-gln-asp-ile-glu-gln-phe-met-ser-cys-ala c) Which sequence looks as if it could form an alpha helix that would participate in a coiled coil structure within a protein? Explain your reasoning.
1. Every third or fourth residue is Leu (heptad repeat motif)
13. Name and briefly describe the three proposed mechanisms of how globular proteins fold in aqueous environments.
1. Hydrophobic collapse model: Hydrophobic residues form the interior of the protein due to the hydrophobic effect, causing a loosely defined tertiary structure called a molten globule. Then, proximal residues in the molten globule interact to form well-ordered secondary and tertiary structures through van Der Waals interactions and hydrogen bonding. 2. Framework model: Initially, local secondary structures form independently. Then, local secondary structures interact to form tertiary structures. 3. Nucleation model: Random interactions lead to a localized region of correct three-dimensional structure, which facilitates the formation of the surrounding tertiary and secondary structures.
8. Explain why secondary structures are so prevalent in proteins.
1. They are formed from combinations of phi and psi angles that minimize steric hindrance. 2. They allow maximum hydrogen bonding interactions in the polypeptide backbone.
14. Answer the following questions based on these four primary protein sequences: 1. Asp-gln-leu-glu-lys-glu-leu-gln-ala-leu-glu-lys-glu-leu-ala 2. phe-gln-ile-asp-met-glu-leu-lys-val-asn-leu-asp-phe-arg-ala 3. ala-gli-tyr-gly-pro-asn-leu-phe-ala-val-ile-lys-asn-cys-ala 4. phe-asn-ser-val-leu-gln-asp-ile-glu-gln-phe-met-ser-cys-ala b) Which sequence looks as if it could form a beta strand, with one surface facing the interior of the protein and the other surface exposed to water? Explain your reasoning.
2. It is the only one that contains alternating hydrophobic and hydrophilic side chains: Ile, Met, Leu, Val, Leu, The alternate with Asp, Glu, Lys, Asn, Asp, Arg.
14. Answer the following questions based on these four primary protein sequences: 1. Asp-gln-leu-glu-lys-glu-leu-gln-ala-leu-glu-lys-glu-leu-ala 2. phe-gln-ile-asp-met-glu-leu-lys-val-asn-leu-asp-phe-arg-ala 3. ala-gli-tyr-gly-pro-asn-leu-phe-ala-val-ile-lys-asn-cys-ala 4. phe-asn-ser-val-leu-gln-asp-ile-glu-gln-phe-met-ser-cys-ala a) Which sequence looks as if it could include a beta turn (reverse turn)? Explain your reasoning.
3. It is the only one with a Gly-Pro sequence for a beta turn.
As temperature increases, proteins are less likely to be in their native, lowest energy conformational state. What is the approximate Tm (melting temperature) for the protein shown?
37 degrees C. The Tm is the temperature at which half the protein is denatured.
14. Answer the following questions based on these four primary protein sequences: 1. Asp-gln-leu-glu-lys-glu-leu-gln-ala-leu-glu-lys-glu-leu-ala 2. phe-gln-ile-asp-met-glu-leu-lys-val-asn-leu-asp-phe-arg-ala 3. ala-gli-tyr-gly-pro-asn-leu-phe-ala-val-ile-lys-asn-cys-ala 4. phe-asn-ser-val-leu-gln-asp-ile-glu-gln-phe-met-ser-cys-ala d) Using the helical wheel drawn in figure 4.34, which sequence looks as if it could form an amphipathic alpha helix? Which amino acids form the hydrophobic and hydrophilic faces of this amphipathic alpha helix.
4. Phe, Val, Leu, Ile, Phe, Met, Ala on the hydrophobic surface, Asn, Ser, Gln, Asp, Glu, Gln, Ser, Cys on the hydrophilic face.
What is a condensation reaction?
A condensation reaction occurs when two molecules join to form a larger molecule and release a smaller molecule(s) in the process. The smaller molecule lost in the reaction is often water, but it can also be methanol, hydrogen chloride, acetic acid or several other molecules.
What is a zwitterion?
A molecule with one negative and one positive end
The GroEL-GroES complex is a(n)...
ATPase and a chaperone. GroEL-GroES is a chaperone that utilizes ATP (thus also an ATPase) to help proteins fold. To function properly, GroEL requires the lid-like co-chaperonin protein complex GroES. ATP binding is required for association of GroES. ATP hydrolysis leads to a conformational change that contributes to chaperone activity.
Which statement best describes the orientation of side chains in α helices and β sheets?
All the side chains of amino acids in α helices are pointed away from the central axis. The backbone atoms fill most of the space within the helix coil. β strands are extended with the side chains of successive amino acids being positioned on alternate sides.
7. Explain how an alpha helix can be amphipathic.
Alpha helices are amphipathic when residues that are hydrophobic (or hydrophilic) are placed 3 to 4 amino acids away from each other. Because there are 3.6 amino acids per turn, amino acids that are three to four amino acids apart will lie on the same side of the helix.
Which secondary structure elements are stabilized by hydrogen bonds?
Alpha helices, beta sheets, parallel beta sheets. NOT random coils. All α helices and all β sheets are stabilized by hydrogen bonds. For the α helices, H-N hydrogen-bond donors and C=O acceptors occur every four residues in the helix. β sheets, whether parallel or antiparallel, are also held together by hydrogen bonds between backbone H-N hydrogen-bond donors and C=O acceptors.
Indicate where the protein secondary structures are found on the Ramachandran plot by dragging each letter to the correct circle in the plot.
As indicated in the figure below, α helices have typical torsion angles of (-70°, -60°) and β sheets have typical torsion angles of (-135°, 135°).
3 ribbon diagram images
Beta strands/sheets: arrows Alpha helices: coils Beta turns and irregular turns that connect these elements: ropes
What is the molecular geometry of the constituents of the peptide plane?
Both the nitrogen and carbon of the peptide plane have three constituents, so they are of the same trigonal planar geometry.
What is the amino acid sequence of the pictured tripeptide, in order from N terminus to C terminus?
By convention, peptide sequences are read from left to right with the amino (or N) terminus on the left and the carboxyl (or C) terminus on the right. The N-terminal amino acid contains a free amino group, while the C-terminal amino acid contains a free carboxyl group. The sequence of this tripeptide is NH3+-Asparagine - Histidine - Arginine-COO-.
3. In the closely packed interior of the tertiary structure of an enzyme, an alanine residue was changed by mutation to a valine, leading to a loss of enzyme activity although that residue was not directly involved in the catalytic function of the enzyme. However, activity was partially regained when an additional mutation at a different position in the primary structure changed an isoleucine residue to a glycine. Based on the structure of the amino acid side chains of alanine, valine, isoleucine, and glycine, explain how the first mutation Ala to Val likely caused a loss of activity, and the second mutation in another region of the protein, Ile to Glycolic, resulted in a partial recovery of enzyme activity.
Changing an Ala to a Val would introduce a bulkier side chain, taking up more volume in the protein interior. The resulting structural adjustments in the tertiary structure must be serious enough to cause the enzyme to lose activity. The replacement of an Ile residue with a Gly allows a tertiary structure close enough to the original structure for partial enzyme activity.
3. Classify all the amino acids into charged, hydrophobic, hydrophilic, and aromatic.
Charged: Glutamate, Aspartate, Histidine, Lysine, Arginine. Hydrophobic: Valine, Glycine, Alanine, Proline, Leucine, Isoleucine, Methionine. Hydrophilic: Serine, Threonine, Cysteine, Asparagine, Glutamine. Aromatic: Phenylalanine, Tyrosine, Tryptophan.
2. Explain how the pKa of an amino acid can differ within a folded protein compared to that of the free amino acid in water.
Chemical properties of nearby fxl groups can alter the pKa of specific amino acids if it is energetically favorable. For example, because positive or negative charges in the hydrophobic interior are energetically unfavorable, the pKa value of a side chain may be altered to favor the neutral state over the charged state at physiological pH.
10. What are the three ways in which quaternary structures can provide increased functionality for a protein?
Complexes can provide structural properties not present in individual subunits and can be a mechanism for regulation of protein function through conformational changes affecting subunit interfaces. Also, bringing functional components into proximity can increase efficiency of biochemical processes.
11. In the small peptide Cys1-Asn-Cys2-Lys-Ala-Pro-Cys3-Ala-Arg-Cys4-Gln-His, there are three possible arrangements of disulfide bonds between two pairs of cysteine residues that will result in two disulfide bonds. One of these combinations corresponds to disulfide bonds between Cys1 and Cys3 and between Cys2 and Cys4. What are the other two combinations of cysteine pairs that would result in two disulfide bonds? To identify the correct arrangement of disulfide bonds, the peptide was treated with the protease trypsin, which generated two oligopeptides under nonreducing conditions that preserve the disulfide bonds. Considering that trypsin cleaves peptide bonds on the C-terminal side of Arg and Lys residues, what is the correct arrangement of disulfide bonds that must exit in the small polypeptide to result in two oligopeptide products?
Cys1-Cys2 and Cys3-Cys4; Cys 1-Cys4 and Cys2-Cys3. Only the Cys1-Cys2 and Cys3-Cys4 combination can explain the observation that the two oligopeptides (with disulfide bonds preserved) are generated by trypsin cleavage; the other two combinations would result in one polypeptide.
Which amino acid is able to form disulfide bonds?
Cysteine The reactive sulfur atom in its side chain enables cysteine to form disulfide bonds.
Which amino acid does NOT have S stereochemistry?
Cysteine (and glycine but that doesn't really count)
Which amino acid has the most reactive side chain?
Cysteine. When 2 cysteine residues are close to each other, they can form a disulfide bond, which helps to stabilize the 3D structures of proteins and form covalent linkages between 2 polypeptide subunits in protein complex.
What is the only L-amino acid in the R configuration?
Cysteine. All other L-amino acids are S.
Due to its partial double bond character, the peptide bond restricts rotation, constraining atoms to lie within the same peptide plane. Rotation in the peptide backbone is around the two bonds that flank adjacent ___ atoms, between which a peptide plane is formed.
Cα. Free rotation in the peptide plane does not occur. Therefore, free rotation is found only around the flanking backbone Cα atoms.
Why is trace mercaptoethanol needed to refold in Anfisen's experiment?
Disulfide exchange. Trace amount is needed to undo any wrong disulfide bonds. -RNAse refolds after removal of denaturing agent. -If it is oxidized in the presence of urea the protein forms incorrect -S-S- bonds, but they find the native structure in the presence of a trace of thiol reagent.
In proteins, only the ____ and _____ that are acids and bases titrate.
Ends of the chain Side chain groups
What did Emil Fischer do?
Founded stereochemistry; origin of L and D nomenclature (wedges and dashes)
8. Briefly explain in terms of the thermodynamics of protein folding why the folded structures of water soluble globular proteins have extensive secondary structure.
Globular proteins will have some polar backbone groups (amide NH and carbonyl O groups) buried inside the protein. When these groups are in the protein's interior, they have lost their favorable hydrogen bond interactions with H2O. The enthalpy change (delta H) for this process would be unfavorable (breaking bonds) if new hydrogen bonds were not made within the protein by secondary structures (hydrogen bonding within alpha helices and between beta strands to form beta sheets).
What type of chaperone is GroEL-GroES?
GroEL-GroES is a chamber-type chaperone. It has multiple GroEL subunits that form a concentric ring with an open center. Multiple GroES subunits form the cap that covers the opening, forming a chamber.
Adding HCl to water changes pH immediately. Adding HCl to sodium acetate gradually changes pH. Why?
HCl to water: The proton (H+) from the acid binds to neutral water molecules to form H3O+ raising the concentration of H+. Happens quickly. HCl to sodium acetate: Sodium acetate is the salt of a weak acid, therefore it has basic character. Neutralizes some of the pH change.
How is proline's structure different from other amino acids?
Has a secondary amine rather than a primary amine.
Thermodynamically, peptide bond ____ is more favorable than peptide bond ____.
Hydrolysis Formation
1. Consider a pentapeptide with the sequence Lys-Tyr-Glu-Asn-His (KYENH). Using the pKa values in table 4.3, make a table to show the approximate charges (if any) on the terminal groups and the side chain of each residue in the peptide at pH 7 and pH 11. What would be the approximate net charge on this pentapeptide at pH 7 and at pH 11?
If amino acid is negatively charged, it can never have a +1 charge, just 0. If it is positively charged, same thing but never -1. If pH < pKa, protonated (more positive) If pH > pKa, deprotonated (more negative)
6. Name the four types of non covalent interactions that stabilize tertiary and quaternary structures of proteins.
Ionic interactions, Hydrogen bonds, van der Waals interactions, and hydrophobic interactions.
Ovalbumin is the major protein constituent of egg white. What is the folded state of ovalbumin in a cooked egg?
Irreversibly denatured. Ovalbumin is irreversibly denatured by excess heat. As the egg white is heated, the temperature is elevated past the Tm of ovalbumin and it denatures (aggregates, firms the egg white, and turns white). However, as the cooked egg is cooled, ovalbumin does not return to its native state (clear and soluble).
1. What is the isoelectric point of a protein? What is the specific term that describes amino acids at this point?
Isoelectric point of a protein: the pH at which it carries no net charge. Zwitterions.
Is histidine always charged?
It is false to presume that Histidine is always protonated at typical pHs. The side chain has a pKa of approximately 6.5, which means that only about 10% of of the species will be protonated. Of course, the precise pKa of an amino acid depends on the local environment.
The charge on a polypeptide is determined by...
Its N and C ends + acid/base side chains
4. What is a peptide bond and how is it formed?
Joins the carboxylic acid group of one amino acid and the amine group of another amino acid; formed by condensation reactions catalyzed by the ribosome. The reaction is unfavorable and requires ATP hydrolysis.
What are the hydrophilic amino acids?
Lysine, arginine, histidine, aspartate, glutamate, serine, threonine, tyrosine, asparagine, glutamine.
If RNase were heated to twice its Tm in the absence of β-mercaptoethanol and then cooled to 4 °C, it would be in what state?
Native state. Excessive heat will denature a protein. Thus, as the temperature is elevated past the Tm, which is the temperature where half of the protein is denatured, more and more protein becomes denatured. However, if the protein can reversibly refold like RNaseA, the protein will return to its native state as the temperature is decreased. Since 4 ºC is well below the Tm for RNaseA, it is mostly folded in its native state.
If the cytoplasm of a cell were to become a much more oxidizing environment, what do you predict would happen to an existing surface-exposed disulfide bond in a typical cytoplasmic protein?
No change. Disulfide bonds already represent the oxidized state of the cysteine side chain. Therefore, there would be no change since the disulfide bond is already oxidized. Further oxidation would require conditions far outside of anything physiological.
Are amino acids and carboxyl groups charged in interior of protein?
No, they cancel.
Levinthal's paradox: do proteins fold by sampling all possible conformations? Provide relational example.
No. It would take longer than the age of the universe to do this. Butane spends 100-1000 fold more time in standard anti conformation than eclipse form. Does this same analysis apply to proteins? Yes.
Which one of the following images represents tertiary structure? Indicate which one by dragging it into the bin labeled Tertiary Structure.
Only the ribbon diagram of the polypeptide (shown below) represents tertiary structure.
Oxidation vs. reduction
Oxidation is the loss of electrons during a reaction by a molecule, atom or ion. Oxidation occurs when the oxidation state of a molecule, atom or ion is increased. The opposite process is called reduction, which occurs when there is a gain of electrons or the oxidation state of an atom, molecule, or ion decreases.
5. Explain the physical relevance of the phi and psi angles.
Phi: The torsional angle between the alpha carbon and the amide nitrogen of an amino acid. Psi: The angle between the alpha carbon and the carbonyl carbon. The values of phi and psi affect the conformation of the peptide backbone.
Composition of proteins
Polymers of monomers, amino acids.
The amino acid sequence determines the structure and function of a protein. The amino acid sequence is called the _________ structure of the protein.
Primary. Primary structure indicates the sequence of amino acids in a protein. It determines secondary, tertiary, and quaternary structure, and hence protein function.
6. Name and briefly describe the four levels of protein structure.
Primary: The specific amino acid sequence of a protein. Secondary: Regular repetitive arrangements of local regions of the polypeptide (alpha helices, beta strands, and beta turns). Tertiary: The spatial location of all atoms in a polypeptide chain. Quaternary: Multi-subunit protein complexes, which can involve multiple copies of the same polypeptide or of different polypeptide chains.
Many diseases, including Alzheimer's disease, are associated with
Protein aggregation. Protein aggregation is the cause of Alzheimer's disease. The amyloid precursor protein is proteolytically processed, which then allows the shorter amyloid β peptide to aggregate.
12. What are the enthalpies and entropic factors that lead to the stabilization of a protein upon protein folding?
Protein folding introduces favorable enthalpies changes because there are more interactions in the folded state than in the unfolded state. These changes result from non-covalent interactions (or disulfide bond formation) because covalent bonds other than disulfide bonds do not change during folding. It is entropically unfavorable because it restricts the number of conformations a polypeptide can take, but the folded protein causes an increase in the disorder of water molecules, which is entropically favorable.
How can cysteine alter protein topology?
Protein topology refers to mutual orientation of regular secondary structures, such as alpha-helices and beta strands in protein structure. The disulfide bond stabilizes the folded form of a protein in several ways: It holds two portions of the protein together, biasing the protein towards the folded topology. That is, the disulfide bond destabilizes the unfolded form of the protein by lowering its entropy.
5 uncharged but hydrophilic amino acids
Serine, threonine, cysteine, asparagine, glutamine. Participate in hydrogen bonding with H2O and have relatively short aliphatic side chains.
What did Anfisen's work show?
Showed convincingly that proteins can indeed adopt their native conformation spontaneously, i.e. sequence determines structure.
It has been observed that in a few cases, the peptide plane is actually bent 4°-5° from being a flat plane. What is the most plausible explanation?
Some other restraint is causing the peptide plane to adopt an unfavorable state. The favorable energetics of the other restraint offset the energetic unfavorability of the skewed peptide plane.
What is the difference between L and D isomer of amino acids?
Stereochemistry term referring to Left handed or Right handed configuration. All our amino acids are L except glycine which has no stereochemistry.
_____ are just as responsible as steric for the stability of the trans amide.
Steroelectronic effects.
Three stop codons
TAG, TGA, TAA
True or False question about amino acids
TRUE: * By definition, all proteins are made of amino acids. * "Molten globule" describes a highly disordered state in the process of protein folding. A protein is initially a random coil, and then quickly collapses to the molten globule state. The protein is still very disordered, but closer to its native state than when a random coil. * Protein folding releases energy. The native state is in a lower energy state than the unfolded conformation. FALSE: * It is not true that unfolded RNaseA cannot be refolded. As one of the first examples of protein folding, Christian Anfinsen showed that RNaseA could be refolded without any help from the cell. * It is not true that all proteins have disulfide bonds. Not all proteins have cysteine residues, so not all form disulfide bonds. Moreover, not all cysteine residues are disulfide-bonded to other cysteines. * It is not true that all proteins require chaperones to properly fold. Some proteins fold spontaneously, like RNaseA.
Which of the following amino acid sequences could form an amphipathic helix?
TVVEAIDRLVDT All the hydrophobic residues lie on one side of the helix.
What level of protein structure describes the spatial location of every atom in a protein?
Tertiary structure (3°) describes the relative spatial location of every atom in a protein. It is determined by the amino acid sequence (1°). It shows the spatial relation of all secondary structure elements and includes the location of other subunits (4°), if present.
What was Anfinsen's hypothesis?
That the native structure of a protein is the thermodynamically stable structure; it depends only on the amino acid sequence and on the conditions of solution, and not on the kinetic folding route.
Given the graph above, calculate the absorbance at 280 nm (UV280) of one mole of the following peptide: Trp-Tyr-Asp-Ala-Trp-Gly-Gly-Lys-Pro
The 280-nm absorbance total comes from two tryptophans and one tyrosine. So, molar extinction coefficient = (Number of Tryptophan residues 5000) + (Number of Tyrosine residues 1000) In this peptide, there are two Trp residues and one Tyr. So, Molar extinction coefficient = 2(5000) + (11000) M-1cm-1 = 11000 M-1cm-1 We know that the molar extinction coefficient can be defined as the absorbance of a one-mole solution at 1 cm path length at a given wavelength. So, the absorbance at 280 nm of one mole of the peptide is 11000 M-1cm-1. Since the aromatic amino acids of proteins absorb 280 nm of light, this is a non-invasive method to determine protein concentration.
If a semi-conservative mutation occurred in the GroEL gene that abolished ATP binding, what functional consequence would you predict?
The GroEL complex would form but would not act as a chaperone. Hydrolysis of ATP is required for the conformational change that imparts chaperone activity.
Which form of cysteine is oxidized?
The disulfide form of cysteine is oxidized (Answer choice B). Remember that the oxidized state has lost electrons (the reduced state has gained electrons.) There are two more electrons (two electrons in each S-H bond) in the reduced form, whereas only two electrons are shared between the S-S bond in the disulfide form.
2. The polarity of the solvent and other environmental factors can affect pKa of a weak acid. Suppose the alpha amino group of a protein has a pKa of about 8 when it is exposed to H2O on the outside of a protein. b) The same alpha amino group in the hydrophobic interior of the protein has the opportunity to form an ionic bond in that hydrophobic environment with a carboxylate group in the side chain of a charged Asp residue. Under these conditions, how would the pKa of this alpha amino group compare with the pKa of the alpha amino group in the hydrophobic interior of the protein without a nearby Asp residue to form this ionic bond?
The pKa for this alpha amino group would increase relative to the example in part a, and would be closer to the pKa of ~8. This ionic bond neutralizes both the NH3+ charge and the COO- charge, reducing the effect of having a charged group in the hydrophobic environment because it is unfavorable to have unbalanced charges.
What is a major consequence of the partial double bond character?
The partial double bond character of the peptide bond greatly reduced rotational freedom, particularly when compared to the φ (phi)/ψ (psi) torsion angles.
Of all the different bond types found in proteins, which has partial double bond character?
The peptide bond has partial double bond character, which is the result of resonance.
9. Consider the two polypeptide strand backbones derived from a large protein that are shown in the illustration that follows. Label the N-terminal to C-terminal direction of each polypeptide strand and determine if these are parallel or antiparallel beta strands. Draw dashed lines to identify the hydrogen bonds that stabilize the beta sheet.
The polypeptide strands are anti-parallel and together form an antiparallel beta sheet stabilized by 2 hydrogen bonds.
If a mutation occurred in which a codon changed to that for proline, what would you expect if the codon were found in a region that formed an α helix?
The proline would disrupt the α helix. Not having the typical H-N bond in the backbone, caused by the cyclization with the side chain, significantly changes the properties of proline. The backbone is much more restricted in terms of favorable torsion angles.
Determine whether the following diagrams depict parallel or antiparallel β sheets.
The relative direction of the polypeptide backbones determines whether β sheets are parallel or antiparallel. Using the figure below, determine the direction of each strand in the β sheet. If they are in the same direction, they are parallel.
Which amino acid is the smallest and least chemically active?
The side chain of glycine is only composed of a single hydrogen, so it is the smallest amino acid.
What is the quaternary structure of a hexamer made of three different homodimers?
There are three different subunits, so the initial designation is ABC. Since there are two of each subunit, adding the numerical designation leads to the final answer of A2B2C2.
Another hexamer is composed of two homotrimers. Using the same letter/number designation as before, indicate its quaternary structure.
There are two different subunits, so the initial designation is AB. Since there are three of each subunit, adding the numerical designation leads to the final answer of A3B3.
Cis vs. trans peptide bonds: which is more stable?
Trans by ~8 kJ mol. Cis less stable due to steric interference; proline residues have ~10% cis conformation.
___ and ___ are the strongest chromophores.
Trp and Tyr. (phenylalanine absorbs, but weakly) UV absorbance at 280 nm is used to determine protein concentration, and fluorescence measurements can be used to study protein structure.
T or F: side chains have overlapping chemical properties.
True. Often even similar residues can't be swapped without changing the fold and function of a protein.
What is the largest amino acid?
Tryptophan, molecular mass 204 D.
13. A homopolymers of lysine residues (polylysine) can adopt an alpha helical conformation or is unfolded, depending on the pH of the solution. Predict whether the conformation of polylysine would be alpha helical or unfolded at pH values of 1, 7, and 11. Explain your reasoning.
Unfolded at pH 1 (the charge repulsion from the numerous positively charged alpha amino groups); mostly unfolded at pH 7 (even though the pKa of the alpha amino groups in the alpha helix would be lower than the normal ~10.8, it would not be as low as pH 7, and therefore still mostly protonated); alpha helical at pH 11 (above the pKa of 10.8, so slightly more than half the alpha amino groups would be deprotonated and uncharged).
12. Explain the roles of urea and beta-mercaptoethanol in Anifisen's experiment son protein folding using the protein ribonuclease. What was the most important conclusion resulting from this experiment that earned Anfinsen the Nobel Prize?
Urea is a denaturing agent. It unfolds proteins by disrupting non covalent interactions; Beta mercaptoethanol is a reducing agent; it breaks disulfide bonds. With sufficient amounts of both urea and beta mercaptoethanol, ribonuclease protein was completely unfolded. The important conclusion was that the primary structure has all the information necessary to specify the tertiary structure.
What are the hydrophobic amino acids?
Valine, leucine, isoleucine, methionine, phenylalanine, proline, glycine, alanine.
Amino acids are polymerized via a _________ reaction.
Water is released as part of the reaction, making it a condensation reaction.
4. Briefly explain how proteins manage to "neutralize" the polarity of main chain carbonyl O and amide NH groups that have been buried in the hydrophobic interior of the protein where the protein folds.
When the amide and carbonyl groups are involved in hydrogen bonding, such as in alpha helix and beta sheet structures, this serves to minimize the effects of these polar groups.
2. The polarity of the solvent and other environmental factors can affect pKa of a weak acid. Suppose the alpha amino group of a protein has a pKa of about 8 when it is exposed to H2O on the outside of a protein. a) Would you expect the pKa to be higher or lower than 8 if the group were buried in the hydrophobic interior of the protein? Explain
a) It is energetically unfavorable for the α amino acid to be charged in the hydrophobic interior, thus the pKa would be lower. The proton can be released, as H3O+, before the amino group enters the interior during the folding process because the strong electrostatic attraction between the lone pair e's in the N atom and the proton is outweighed by the unfavorable condition of a charge in a hydrophobic environment.
10. The ionization of the amino acid by three weak acid groups with kp1 = 2.1, pk2 = 4.1, and pk3 = 9.9. The following illustration shows four possible ionic forms of Flu that might occur in solution and a titration curve for Glu. Which of these ionic forms represents the zwihterion of Glu? Identify the structure by letter that corresponds to the ionic form of Glu that predominates at each pH value indicated by the arrows. If two ionic forms are present equally, select two choices.
a, a+c, d, d+b, b. Zwitterion of cysteine is C.
7. Answer the following on the basis of the tetra peptide structure shown here. a) Name the residues in the peptide. b) Draw boxes to identify the peptide bonds between residues 1 and 2 and between residues 3 and 4. c) Draw a rectangle to identify the six atoms that are coplanar with the peptide bond between residues 2 and 3. d) Predict a pH range that would result in the ionization state of this tetra peptide.
d) Based on the pKa values of for the three ionizable residues + N and C terminal, a pH of 11-12 would result in the charges shown.
All linear proteins have a ____ where their net charge is zero.
pI = pH
From the plot of torsion angles shown, is this protein mostly composed of α helices or β sheets?
α helices. As indicated in the figure below, α helices have typical torsion angles of (-70°, -60°) and β sheets have typical torsion angles of (-135°, 135°).
