B1_MidtermExam1_2023_QUESTIONS_CompleteKey_updated101323

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Page 1 of 10 BIOCHEMISTRY I (CHEM-UA 881) Fall 2023 MIDTERM EXAM 1, Oct. 6, 2023 One point extra credit for completing the 3 lines below properly NAME : ___________________________ N-NUMBER : __________________________ TA & RECITATION TIME : __________________________ PLEASE WRITE LEGIBLY & BE CONCISE. Your answers must be your own! No calculators, no use of electronic devices, no communication between students. Partial credit will be given. (There are a total of 9 pages in the exam, including this page) USEFUL INFORMATION: Group Typical pKa Terminal a -carboxyl group 3 Aspartic acid/Glutamic acid 4 Histidine 6 Terminal a -amino group 8 Cysteine 8 Tyrosine 10 Lysine 11 Arginine 12 USEFUL EQUATION: pH = pKa + log([A - ]/[HA]) QUESTION PTS YOUR SCORE 1 30 2 25 3 10 4 10 5 15 6 10 TOTAL 100

Page 2 of 10 1. Multiple Choice. Circle the correct answer for each. ( 3 points each; 30 total ) i. Which of the following statements is TRUE about Anfinsen’s famous folding experiments on RNAse A? a. Anfinsen concluded that the primary sequence of a protein alone is sufficient to determine structure. b. Formation of protein disulfide bonds alone was enough to restore the activity of RNAse A. c. Addition of urea returned RNAse A to its native folded and active state. d. Disulfide bonds in RNAse A could be formed with the addition of beta- mercaptoethanol. ii. Cystic Fibrosis can be considered a protein folding disease associated with an amino acid mutation. What best describes the effect of the altered protein folding? a. Gain of function b. Loss of function c. Cooperative function d. None of the above iii. What is the characteristic sequence of the alpha-helical keratin protein? (Hyp= hydroxyproline) a. Repeating (Gly-Pro-Pro) b. Repeating (Gly-Hyp-Pro) c. Repeating (Gly-Pro-Hyp) d. Repeating (Gly-Gly-Hyp) e. None of the above iv. Disulfide bonds represent important ____________ that influence protein tertiary structure stability. a. non-covalent interactions b. van der Waals interactions c. hydrogen bonds d. salt bridges e. covalent bonds v. On average, what percentage of lysine residues in a peptide are present in the charged form at pH = 8? a. 99% (actually 99.9%) b. 90% c. 50% d. 10% e. 1%

Page 3 of 10 vi. Which of the following non-covalent interactions is driven primarily by changes in entropy ( D S)? a. Hydrogen bonding b. Hydrophobic effect c. Cation-pi interactions d. van der Waals interactions e. Electrostatic interactions vii. In a protein structure, which amino acid is the most likely to exhibit variation in the omega dihedral angle? a. Tryptophan b. Cysteine c. Glycine d. Proline e. Arginine viii. Which of the following amino acid residues would most likely be found in the interior of a water-soluble, globular protein? a. Glu b. Asp c. Lys d. Arg e. Ile ix. Which of the following proteins does not represent a fibrous protein? a. Silk fibroin b. Keratin c. Myoglobin d. Collagen e. None of the above

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Page 4 of 10 x. In recitation, we discussed dihedral angle determination using the following amino acid depiction: Which of the following is true about the dihedral angle represented by atoms 1-4? (1 = N, 2 = C, 3= C, 4= N) a. The angle is f (“phi”), and it equals 0 ° b. The angle is f (“phi”), and it equals 180 ° c. The angle is y (“psi”), and it equals 0 ° d. The angle is y (“psi”), and it equals 180 ° e. The angle is w (“omega”), and it equals 180 ° 2 1 3 4 N N C C O

Page 5 of 10 Part II: Short answer . Please provide a brief response to each question (5 pts each). 2A. List three of the techniques that we discussed in lecture that are used to determine the three-dimensional structure of a protein at atomic resolution. X-ray crystallography NMR spectroscopy Cryoelectron microscopy 2B. A titration curve for the individual amino acid Alanine is shown below. Draw the structure of Alanine at point A, showing stereochemistry and the proper charge state. (Note this is for the free amino acid, not as part of a peptide.) H 3 N O - O

Page 6 of 10 2C. In the space below, draw a cartoon representation of two parallel b -strands. Do you think the strands be joined by a b -turn secondary structure? Explain briefly. The N-termini are at the bottom of the arrow. The C-termini are at the head of the arrow. (this is implied by the depiction of the strands) No, the N-terminus and C-terminus of beta-strands in a parallel beta-sheet are not in proximity, so they cannot be joined by a short beta-turn. Beta-turns are typically 3 or 4 residues in length, not sufficient to make this kind of connection. 2D. Both the Alzheimer’s beta-peptide and prion proteins can form amyloid deposits that are characteristic of many neurodegenerative disorders. For both these proteins, a conversion from a soluble form to an amyloid form is associated with a conformational rearrangement. Describe the general feature of this conformational rearrangement. Why does this allow formation of amyloid? Both proteins undergo a rearrangement from alpha-helix to beta-strand (sheet) structure as an early step in amyloid formation. This is because amyloid is formed by propagating inter-molecular beta-sheet structures, which require the presence of beta-strands. Hence, the important of the conformational rearrangement from alpha-helix to beta- strand. The phrase “conformational rearrangement” generally refers to a significant structural alteration within the protein backbone. Typically, this occurs through an intramolecular change in secondary structural features. Many students correctly noted that in the formation of amyloid, individual beta-strands or small beta-sheets can form intermolecular associations to create larger beta-sheets, solenoids, fibrils, etc. Although this may not usually be described as a conformational rearrangement, it is an alteration in (quaternary) structure features. These answers received large partial credit.

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Page 7 of 10 2E. Describe very briefly the function of the Hsp70 and GroEL/GroES protein systems. Which of these molecular chaperones/chaperonins rely on ATP hydrolysis for their function? Both protein systems help fold nascent proteins or refold improperly folded proteins. Both rely on ATP hydrolysis. Part III. Free response/Data Analysis. Please respond to each question in the space provided. 3. You are studying a peptide with the following amino acid sequence (10 pts): PRIME a) In the space below, draw the chemical structure of your peptide at pH = 9.0 , including correct charges on side chains and the groups at the termini. Show stereochemistry along the backbone. 6 points b) What is the net charge on the peptide at pH = 9.0 ? 4 points -1 4 . You are purifying a protein that forms a homodimer quaternary structure stabilized by a disulfide cystine crosslink. You are using size exclusion chromatography, and you observe that the protein elutes (flows out of the bottom of the column) about 1 hour after loading on the top of the column. You then repeat the purification in the presence of beta- mercaptoethanol. Do you expect the protein to elute faster, or more slowly, or at the same rate? Justify your answer using a description of the matrix (beads) that are used to perform size exclusion chromatography. (10 pts) The protein should elute more slowly. This is because the beta-mercaptoethanol has destroyed the cystine crosslink of the dimers, creating protein monomers. This protein will be smaller in size than the protein dimer. The smaller protein will be able to penetrate the porous core of the size exclusion chromatography beads, which will cause the proteins to take a longer time to pass through the column. N H H N N H H N N H O - O O O O O HN H 2 N NH 2 + S O - O

Page 8 of 10 5. Assume that the following amino acid sequence forms an a -helix (15 pts): AFHAYSEMASALD a) Draw a helical wheel representation of the structure. 6 points b) Is the structure amphiphilic? Explain your response very briefly. 3 points Yes. Note that residues 3,6,7,10, and 13 form a polar face. While residues 1,2,4,5,8,9,11 and 12 form a hydrophobic face. c) Which two amino acids will form a favorable non-covalent interaction at pH = 5? 3 points Histidine 3 and Glutamate 7. They are in proximity to form a salt bridge. Note that the Aspartate is too far away to make a similar contact. d) Estimate the distance (in Å) between the side chains from part C within the a -helix. 3 points 5.4 A. His 3 and Glu 7 are one helical turn away from each other. The alpha helix is 5.4 Angstroms per turn.

Page 9 of 10 6. Examine the structure of the two proteins labeled (a) and (b) below and the Ramachandran plots for these proteins (10 pts). a) Which one of the four common types of protein folds is seen in protein (a)? Which one is seen in (b)? 3 points a: alpha/beta (mixed alpha + beta is acceptable for full credit) b: all alpha b) Ramachandran plots show variations in the phi and psi dihedral angles but do not bother to show the omega (amide) dihedral angle. Why? 4 points Peptide bonds have a double bond character, and are typically trans in natural proteins, such that omega is 180 ° (although some amino acids can form cis peptide bonds in proteins, but this is less common). Because there is little variation, there is no reason to include this angle in the plots. c) Which of the two Ramachandran plots, labeled c and d above, is more likely to be derived from protein a ? Which plot is derived from protein b ? Briefly explain your answer. 3 points The protein in (a) has primarily b -structure (represented by flat arrows) with a small amount of a -helical structure (represented by coils). The protein in (b) has only a -helical structure. In lecture, we discussed that b -structures fall in the upper-left quadrant of the Ramachandran plot. Plot (c) has many points in the upper-left quadrant, whereas part ( d ) has few; so the plot that corresponds to (a) would be (c) . Right-handed a -helices have dihedral angle values that put it into the lower-left quadrant of the Ramachandran plot, so protein (b) must correspond to graph (d) .

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