Biochem Fall 2023 Practice Exam 1_KEY

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Practice Exam I KEY Biochemistry 1 Fall 2023 Exam 1 will cover topics from Lectures 1-8. This practice exam is longer than the actual Midterm. Please use the pKa values in your lecture notes. Part I: Multiple Choice Questions. Please provide one answer for each of the following multiple choice questions. If more than one answer is selected, the question will be marked as incorrect. 1. Which of the following molecules would you expect to form a hydrogen bond with water? a. CaCl 2 b. NH 3 c. CH 4 d. NaCl 2. If the equilibrium constant ( K eq) is greater than 1, what is the value of Δ G °? a. Δ G ° > 0 b. Δ G ° = 0 c. Δ G ° < 0 d. Δ G ° > 1 3. Which of the following statements is true about α helices? a. The center of the helix is an open channel. b. There are about seven amino acids per helical turn. c. The amide backbone dipoles are aligned in the same direction. d. The helical backbone structure is stabilized by ionic interactions. 4. Which three amino acids form the repeating unit of collagen? a. Proline-Serine-Glycine b. Hydroxyproline-Proline-Alanine c. Serine-Proline-Hydroxyproline d. Glycine-Proline-Hydroxyproline 5. Which type of chromatography separates proteins using specific binding properties? a. affinity chromatography b. gel filtration chromatography c. size-exclusion chromatography d. high-performance liquid chromatography 6. Protein NMR is more useful than X-ray crystallography for studying a. secondary structure elements. b. large proteins. c. protein unfolding. d. static protein structures.
7. Which of the following amino acid residues would most likely be found on the surface of a water-soluble, globular protein? a. Phe b. Ser c. Leu d. Trp 8. The circled atoms in the dipeptide below describe which dihedral angle? a. f dihedral, “phi” b. y dihedral, “psi” c. w dihedral, “omega” d. none of these 9. The structure below depicts a ____________ -sheet? (Circle one) Antiparallel b -Sheet Or Parallel b -Sheet H 2 N H N R 1 O R 2 OH O
10. We discussed how the energetics of protein folding can be driven by several different processes that affect the enthalpy or entropy of the system. Which of the following statements does NOT accurately describe a process that significantly influences the direction of protein folding? a. conformational change of the protein chain, leading to a loss of entropy b. noncovalent interactions between side chains established by folding (H-bonds, van der Waal interactions, etc), which are enthalpically favorable c. the hydrophobic effect, which describes non-covalent interactions between non-polar side chains that contributes favorably to the enthalpy term d. the hydrophobic effect, which describes the increased entropy of water molecules in the system as hydrophobic collapse occurs in the protein interior
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Part II: Short answer . Please write a brief answer in response to each question. 1. Many different proteins can form amyloid deposits, contributing to protein folding diseases. Explain why such a variety of different protein sequences can all form the same type of protein structure and why these assemblies are so stable. Amyloid is formed when protein molecules associate through beta strands to form extended beta- sheets, that can be extended to include many molecules and form fibrous assemblies. Because the critical interactions that define these structures are between the polypeptide backbone (see figure), a variety of different proteins with different side chain sequences can form amyloid. The density of hydrogen bonding, and the extended H-bond network establish the stability of the assemblies. 2. The majority of protein structures have been determined using either NMR (nuclear magnetic resonance) or X-ray crystallography. Describe an additional technique that has recently been used to determine protein structures, often at atomic resolution? Cryo-electron microscopy has also been used to determine protein structures. This technique uses a focused beam of electrons to provide a series of images of proteins (or protein assemblies) that are immobilized in a thin film of vitreous ice. The pattern of electron scattering is detected and used to generate a set of two-dimensional images. From these data, image reconstruction techniques can be used to recreate the three-dimensional structure. 3. What is the role of ATP hydrolysis in the GroEL/GroES chaperonin? ATP is hydrolyzed by GroEL protein subunits, driving a conformational change in the protein that enlarges the chamber formed by the quaternary structure. This alteration helps an improperly folded protein within the chamber to refold.
4. Clearly label the a -helix and b -strand regions on the Ramachandran Plot below. 5. Draw a generic hexapeptide backbone with the pattern of hydrogen bonding in a protein a - helix indicated with arrows. Side chains can be indicated as R and the correct stereochemistry at the a -C should be shown. 6. The correct folding of ribonuclease to its active three-dimensional structure is determined, in part, by the proper pattern of cystine disulfide crosslinks. Provide an arrow pushing mechanism for disulfide scrambling for the reaction below. H 2 N N H H N O O O N H H N O O N H OH O R R R R R R S S HS S S SH b -strand a -helix d
7. For the two peptide sequences listed, answer the questions below: Peptide 1: IHATEYANKEESH Peptide 2: RARELIPSTICK (a) Would you expect an ionic interaction between peptide 1 and 2 at pH = 8? Explain by calculating total peptide charges as part of your answer. YES: At pH = 8, peptide 1 is -2.5, peptide 2 is +1.5 (b) Considering your answer for the total charge on peptide 1 above, please circle the resin that you would use for ion exchange purification at pH=8. Circle one. R S S R R SH Base R S R S S R + R S O O - N H
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Part III. Free response/Data Analysis. Please respond to each question in the space provided. 8. Draw the chemical structure of the peptide PCHEM at pH = 7, including the groups at the termini. Please show correct stereochemistry along the backbone. 9. What is the net charge (+, 0, -) of the indicated free amino acids at the given pH values (Use pKa values in your lecture notes). GLYCINE pH 2 + pH 7 0 pH 12 ASPARTIC ACID pH 2 + pH 7 pH 12 ARGININE pH 2 + pH 7 + pH 12 ~ 0 (-.5) 10. In contrast to ribonuclease, some proteins cannot reach their native state without the assistance of proteins known as molecular chaperones. This means the thermodynamic hypothesis of protein folding does not apply to these proteins. True or False? Explain your reasoning. FALSE. - some proteins require molecular chaperones to reach their native state, which is their global free energy minimum H 2 + N N H H N O O O N H H N O O O SH O O S N H N
-Chaperones accomplish this by allowing misfolded proteins trapped in a local free energy minimum (or kinetic trap) to refold, thus allowing them to reach their native state 11. An enzyme (MW 24 kDa, pI 5.5) is contaminated with two other proteins, one with a similar molecular mass and a pI of 7.0, while the other has a molecular mass of 100 kDa and a pI of 5.4. Suggest a protein purification procedure, based on “CHASM” properties, to efficiently purify the contaminated enzyme on a preparative scale. - First do an ion-exchange column to separate out enzyme from the protein with a pI of 7.0. - Then do a size-exclusion (also called gel-filtration) column to separate out enzyme from the protein with a 100kDa mass. This also separates our target protein from the salt present in the elution step done to remove our enzyme from the ion-exchange column. *Doing the ion-exchange second gives us our sample in a high salt concentration solution because the elution is performed by increasing the salt concentration. *2D SDS PAGE is not an acceptable answer because this would not achieve a preparative scale purification of the enzyme (this is an analytical technique). 12. Lysine has a relatively high propensity to form an a -helix. Ornithine, which is shorter by one methylene group, is found to have a much lower helix propensity. Based on your knowledge of secondary structures, account for this observation. (Hint: Glutamic acid also has a stronger helical propensity than aspartic acid and for the same reason as lysine versus ornithine.) H 2 N OH O NH 2 H 2 N OH O NH 2 lysine ornithine
Ornithine has low helical propensity because its side chain functional group can hydrogen bond with the backbone carbonyl (as shown). The ornithine hydrogen bonding competes with the typical i à i+4 hydrogen bond observed in alpha helices. Lysine side chain is longer and makes less contact with the backbone. The same reasoning applies to aspartic acid (and asparagine) versus glutamic acid (and glutamine) propensities. 13. An enterprising NYU biochemistry student decided to check how substitution of different residues impacts helicity of a polyalanine sequence. This student devised an experiment such that the i and i+4 residues in the sequence are changed while the rest of the sequence is kept intact. Predict the impact of the substitutions on each sequence and rationalize your predictions. Parent sequence: AAAAAAAAA i. AAVAAAVAA Circle one: Higher helicity than (A) 9 or Lower helicity than (A) 9 Rationale for your choice: Valine is branched and has a low propensity to form an alpha helix. ii. AAGAAAGAA Circle one: Higher helicity than (A) 9 or Lower helicity than (A) 9 Rationale for your choice: Glycine has no side chain – it is highly flexible and is therefore regarded as a “helix-breaker”. iii. AARAAAWAA Circle one: Higher helicity than (A) 9 or Lower helicity than (A) 9 Rationale for your choice: The arginine and tryptophan side chains are positioned in proximity on the exterior of the helix, separated by one helical turn. The side chains can interact to allow a stabilizing cation/pi non- covalent interaction. iv. AARAAARAA Circle one: Higher helicity than (A) 9 or Lower helicity than (A) 9 N H O N H N O O HN O R R R R N H O N H N O N O O HN O R R R H H H α -helix 1 13 11 9 7 5 3 N H O N H N O O HN O R R R R N H O N H N O N O O HN O R R H H H NH H ornithine N H O N H N O O HN O R R R R N H O N H N O N O O HN O R R H H H NH H ornithine
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Rationale for your choice: The arginine side chains are positioned in proximity on the exterior of the helix, separated by one helical turn. The side chains can interact to create a destabilizing electrostatic repulsion. v. AAEAAARAA Circle one: Higher helicity than (A) 9 or Lower helicity than (A) 9 Rationale for your choice: The arginine and glutamate side chains are positioned in proximity on the exterior of the helix, separated by one helical turn. The side chains can interact to create a stabilizing electrostatic interaction. 14. (Miesfeld, Chapter 5, question 31) A functional assay was designed to isolate a putative wound- healing growth factor present in plant extracts made from an exotic flower found high in the forest canopy of the Amazon rain forest. Initial field studies of the purified factor identified a tetradecapeptide (14 amino acid residues) that consisted of 2 mol of glycine and 1 mol of each of 12 other amino acids—none of which was alanine, histidine, leucine, serine, threonine, tryptophan, or valine on the basis of available amino acid standards. Although the field laboratory was set up to use Edman degradation sequencing to deduce partial amino acid sequences, the efficiency of the reaction limited its application to tetrapeptides and smaller. Therefore, the Edman degradation data were supplemented by a combination of proteolytic cleavage assays with trypsin, chymotrypsin, and V-8 protease, as well as chemical cleavage with cyanogen bromide. Using the following information collected by the field biochemist, determine the most likely sequence of the tetradecapeptide, written left to right from the N-terminal residue using the single- letter amino acid code. 1. Cleavage with trypsin yielded a hexapeptide, a septapeptide and a single amino acid that was identified with amino acid standards as glutamine. 2. Cleavage with chymotrypsin yielded a hexapeptide, a pentapeptide, and a tripeptide with the amino acid sequence G-I-F. 3. Cleavage with V-8 protease yielded a heptapeptide, a tripeptide with the sequence P-R-Q, and a tetrapeptide with the sequence G-Y-N-D. 4. Cyanogen bromide chemical cleavage yielded a decapeptide and a tetrapeptide with the sequence G-I-F-M. Trypsin cleaves on the carboxyl-terminal side of lysine or arginine residues. Because the 1st or 14th amino acid must be Q (it cannot be the 7th or 8th amino acid based on the substrate specificity of trypsin), the 6th or 7th amino acid and the 13th or 14th amino acid must be K or R. However, because chymotrypsin cleaves on the carboxyl side of tyrosine, tryptophan, and phenylalanine residues and generated the tripeptide G-I-F, and cyanogen bromide cleaves on the carboxyl side of methionine and generated the tetrapeptide G-I- F-M, the N-terminal tetrapeptide sequence must be G1-F2-I3-M4, and the C-terminal amino acid must be Q14. Using the data collected from the V-8 protease cleavage analysis (V-8 cleaves on the carboxyl side of glutamate and aspartate), the C-terminal tripeptide must be P12-R13-Q14, which means the sixth amino acid must be K6, as only G is represented twice in the
tetradecapeptide. In addition, the tetrapeptide produced by V-8 cleavage must be adjacent to this C-terminal tripeptide and correspond to the amino acid residues G8-Y9-N10-D11. Finally, the heptapeptide generated by V-8 protease must correspond to the first seven amino acids and have the sequence G1-F2-I3-M4-X5-K6-E7, in which X5 must correspond to cysteine (C5) as this is the only amino acid not yet accounted for based on the initial amino acid composition analysis. The illustration below summarizes these findings.