Biochemistry I Exam 2 Answer Key October

Page 1 Exam II Biochemistry I I. _________________/30 II. _________________/45 III. _________________/25 TOTAL ________________/100 I. MULTIPLE CHOICE. (30 points; 3 pts each) Choose the BEST answer to the question by circling the appropriate letter. 1. A D-amino acid would interrupt a b-strand made of L-amino acids. Another naturally occurring constraint on the formation of a b-strand is the presence of: A. B. C. D. E. 2. Which of the following is not considered one of the four major levels of protein structure? A. B. C. D. E. 3. quaternary structure primary amino acid sequence domain structure three-dimensional conformation of entire polypeptide secondary structure Which statement about 3° structure determination is false ? A. B. C. D. E. 4. a negatively charged Arg residue. a positively charged Lys residue. two Gly residues side by side. a nonpolar residue near the carboxyl terminus. a Pro residue. In X-ray crystallography, a structure determined at 2 Å resolution has more detail than one determined at 5 Å resolution. In NMR, COSY is able to determine bond angles. Some of the structure could be conferred by crystal packing. NMR structures are generally at higher resolution than X-ray structures, yet they reveal details of conformational flexibility. NMR can readily distinguish between Thr and Val, whereas X-ray crystallography cannot. The concept of "induced fit" refers to the fact that: A. B. C. D. E. when a substrate binds to an enzyme, the enzyme induces a loss of water (desolvation) from the substrate. substrate binding may induce a conformational change in the enzyme, which then brings catalytic groups into proper orientation. enzyme-substrate binding induces an increase in the reaction entropy, thereby catalyzing the reaction. enzyme specificity is induced by enzyme-substrate binding. enzyme-substrate binding induces changes in the substrate towards the transition state. Page 2 5. The major driving forces involved with the formation of very large multisubunit protein complexes, for example the protein capsid of a virus, are: A. B. C. D. E. 6. Which of the following statements is false ? A. B. C. D. E. 7. are found on the outside of the helix spiral. generate the hydrogen bonds that form the helix. stack within the interior of the helix. cause only right-handed helices to form. alternate between the outside and the inside of the helix. Amino acid residues commonly found in type I and/or type II b-turns are: A. B. C. D. E. 10. conjugate the protein to a known molecule. determine its amino acid composition. determine its molecular weight. determine its amino acid sequence. purify the protein. In an a helix, the R groups on the amino acid residues: A. B. C. D. E. 9. Each strand of the protein chain in collagen is a left-handed helix. For S Æ P, an enzymne catalyst will always increase the reaction rate. At the end of an enzyme-catalyzed reaction, the functional enzyme becomes available to catalyze the reaction again. Substrate binds to an enzyme's active site. Collagen is an abundant globular protein in mammalian organisms. For the study of a protein in detail, an effort is usually made to first: A. B. C. D. E. 8. free energies of hydrolysis of such molecules as ATP and GTP. the same weak, noncovalent interactions that determine the native conformation of a single polypeptide. the free energies of binding by small ligands. entropy increases resulting from the formation of many new peptide bonds. entropy decreases resulting from the formation of symmetric crystalline shapes, for example, icosahedrons. Ala and Gly. Pro and Gly. two Cys. hydrophobic. those with ionized R groups. Pauling and Corey's studies of the peptide bond showed that: A. B. C. D. E. for a protein in solution at pH 7, any one of a large number of conformations is equally probable. the primary structure of all proteins is very similar, although the secondary and tertiary structure may differ greatly. the peptide bonds in proteins are very unusual, bearing almost no resemblance to peptide bonds in small model compounds. the peptide bond is essentially planar, with no rotation about the C-N axis of the amide bond. the structure of a peptide bond is so complex that even Pauling could not understand it. Page 3 II. SHORT ANSWER. (45 points) Give a brief answer or diagram for each problem or question below. 11. In the lab, when you use BSA for making a standard curve for a colorimetric assay, what substance are you trying to measure? What quantity is this assay meant to determine? What assumptions are you making relative to an unknown (not BSA) you are measuring using that standard curve? (3 pts) 12. What was the purpose of heating the BSA prior to digestion with trypsin in the Experiment in Chapter 2 of the laboratory? Would not heating have affected the results? How? (2 pts) 13. In lab, you used histidine with its 3 dissociable protons: one with a pK a of 2.3, one with a pK a of 6.5, and the third with a pK a of 9.7. Sketch a properly labeled titration curve for histidine (HHis+) titrated with NaOH; indicate where on the curves the pH = pK a , and indicate the region(s) of the curve in which the highest buffering occurs. (3 pts) 14. Diagram a peptide bond from one Ca to the next (no R-groups please) in the resonance form that best explains its characteristics. For the Ca -Cpeptide and the Npeptide-Ca bonds, label each by the name of the angle that describes its conformation. (3 pts) Page 4 15. A biochemist purified a new enzyme and subjected a sample to SDS-PAGE (SDSpolyacrylamide gel electrophoresis). The results showed that there were three different proteins present with relative molecular weights of 20 kDa, 25 kDa, and 40 kDa. When sedimentation equilibrium ultracentrifugation was performed on the preparation, a molecular weight of 130 kDa was determined. What is the quaternary structure of this enzyme? Use proper nomenclature. Diagram the expected intensities of subunits on a stained SDS-PAGel. (5 pts) 16. The X-ray crystallographic analysis of proteins often fails to reveal the positions of parts of the peptide chain, particularly the N- and C-terminal few amino acids. Explain. (2 pts) 17. Describe the primary, secondary/tertiary, and quaternary structure of collagen. How is pro-collagen different from collagen? (5 pts) 18. In the process called ________, phenylisothiocyanate (below) reacts with Nterminal amino group of a polypeptide, ultimately cleaving the first peptide bond releasing a phenylthiohydantoin derivative of the N-terminal amino acid. (2 pts) 19. How would the following agents or procedures interfere with or disrupt the different levels of protein architecture? Why? (3 pts) a) Addition of SDS b) Oxidation of cysteine with performic acid to cleave disulfide bonds c) Addition of proteases such as trypsin Page 5 20. You purified a 20 kDa protein and determined its mass spectrum by ESI-MS and got the data shown below. Circle the letters for the following statements about the data if they are true? (the numbers above each peak are the m/z for that peak) (3 pts) a. you must have more than one protein in the sample because there is more than one peak in the spectrum. b. the peaks with a higher m/z are more charged than those with the lower m/z. c. the peak at m/z = 1001.0 has a z = +20 d. the protein has a mass of 1001.0 Da, not 20 kDa as you had first thought. e. you cannot interpret this mass spectrum without knowning the amino acid composition. 21. Several biotechnology/pharmaceutical companies have explored the idea of using "stapled" peptides for stabilizing small therapeutic peptides as ahelices. Unnatural amino acids with reactive side-chains are introduced into these peptides and then induced to covalenlty cross-link to form the "moleculaar staple," which theoretically stabilizes the a-helical conformation of the peptide (see below). a. For the amino acids X and Y to form the cross-link shown above, how far apart in the primary structure of the peptide must they be? Briefly, explain your answer. (2 pts) b. Before and after the stapling reaction, the circular dichroism spectra were determined (see below). Which spectra belongs to the unstapled and which belongs to the stapled peptide, and why. Was this idea successful? (2 pts) Page 6 22. Your Edman Degradation sequenator was broken and there is a postal strike so you could not send your peptide for sequence determination. You decide to use chemical techniques. This decapeptide had an amino acid composition: (Gly)3, (Phe)2, Pro, Arg, Met, Leu, Ala. A. 1) Reacting the native peptide with FDNB followed by complete hydrolysis (N-terminal analysis) released 2,4-dinitrophenylglycine. 2) Incubation with CNBr yielded two fragments, a tetrapeptide with composition (Gly)2, homoserine lactone, and Phe, and a hexapeptide. The tetrapeptide yielded 2,4-dinitrophenylglycine, and the hexapeptide yielded 2,4-dinitrophenylleucine after N-terminal analysis with FDNB. 3) Proteolytic cleavage of the native decapeptide by trypsin gave two fragments, a hexapeptide and a tetrapeptide; N-terminal analysis of the tetrapeptide yielded 2,4-dinitrophenyl-phenylalanine. 4) Chymotrypsin cleavage of the native peptide gave a tripeptide composed only of glycine and Phe, and a heptapeptide. B. The information in A) should yield the sequence of all but the C-terminal residues. You decide to use your brand new tandem MS/MS instrument. You perform MS/MS on the C-terminal tryptic tetrapeptide to determine the sequence of this remaining peptide. The pattern gives you MW fragments of 444, 369, 280, 279, 165, 164, 75. [NOTE: MW of tetrapeptide is 444 and MW of Ala, Gly, Phe, and Pro are 89, 75, 165, and 115, respectively. Phe-Pro has MW of 280.] Put the sequence based on this information in the spaces below. (10 pts) ____ ____ ____ ____ ____ ____ ____ ____ ____ ____ III. 23. ________1. ________2. ________3. ________4. MATCHING. (25 points) Match the types of protein structure on the right with techniques for structure determination for which they can be determined effectively. In the blank to the right of number 1, cite an example of a type of structure that corresponds to your answer. (5 pts) Circular Dichroism _____________________ Diagonal Peptide Mapping Edman Degradation Gel filtration & SDS-PAGE 24. A. B. C. D. E. primary structure tertiary structure secondary structure quaternary structure disulfide bond structure For each EC enzyme category or protein sequence determination method on the left, match to the most appropriate enzyme with a trivial name or description of the method on the right. (8 pts) _________1. _________2. _________3. _________4. _________5. _________6. _________7. _________8. oxidoreductase transferase hydrolase isomerase ligase amino acid analysis diagonal peptide mapping reduction and alkylation A. B. C. D. E. F. G. H. Tyrosine transaminase determination of disulfide bond formation determination of amino acid content trypsin Tyrosine-tRNA aminoacyl synthetase blocking disulfide bond formation lactate dehydrogenase phosphoglucomutase Page 7 25. Match the items on the left _________1. Trivial enzyme name (only) _________2. Systematic enzyme name and EC number _________3. sequential random bi bi with the BEST item for each on the right: (5 pts) A. Kd _________4. D. Enzyme proficiency B. k cat A C. B P E E A B P Q FAB E B A _________5. Enzyme turnover rate Q A E. P E E Q B P Q E F. k cat/k uncat G. H. I. J. kinase isomerase EC 1.2.1.32 a lyase EC 1.2.1.32 an oxidoreductase From the types of bonds and interactions on the right, identify which is most responsible for the structures described on the left. If there are two types of bonds of nearly equal responsibility, identify both. There are no more than two for each. (7 points) _________1. a tight fit (≤2Å) of complementary shapes A. ionic interactions of a ligand to its receptor _________2. secondary structure of proteins B. hydrophobic interactions _________3. the triple helix of collagen C. van der Waals interactions _________4. the compactness of the interior of D. covalent bonds myoglobin _________5. the association of collagen molecules to E. hydrogen bonds make collogen fibrils _________6. the association of hemoglobin subunits in its quaternary structure 26. Page 1 No. on Test Correct Answer 1 2 3 4 5 6 7 8 9 10 11 12 E C D B B E E A B D You are measuring PROTEIN. You are quantifying protein concentration or amount of protein. You assume that BSA reacts to give a color in a similar way as what you are measuring. 1. Heating denatured the BSA, thus making all the Arg and Lys as accessible to trypsin as possible. 2. Without heat the number of these residues measured would likely have been fewer due to some being buried or at least the reaction taking longer to reach completion. 13 -The flatest part the pH = pK a and are the best buffering portions of the curve. pH 9.7 6.5 2.3 1 2 OH- Equivalents -14 3 Page 2 No. on Test Correct Answer 15 Since there must be at least one of each subunit, the MW would be ≥85 kDa. Sedimentation equilibium ultracentrifugation measured the native MW of 130 kDa, therefore the only combination that gives 130 would be 45 kDa more, so there must be two each of the smaller subuinnts (20x2 + 25x2 + 40 = 130). So the quaternary structure would be ab2 g 2 , where either the 20 or 25 kDa subunits are b or g and the 40 kDa subunit is a. Make sure each subunit is defined. There will be three bands 20 kDa will be about equal intensity with the 40 kDa due to half the size, but double the number with the 25 kDa with slightly more intensity than the 20 kDa. 16 In a protein crystal, the residues at the end of a polypeptide chain, and some interior residues, may experience fewer intramolecular contacts or be innately mobile. They tend to not occupy a set position in the crystal (less ordered, more mobile). This disorder prevents them from generating a coherent diffraction pattern when the x-ray beam hits these parts of the protein and without a diffraction pattern the electron density cannot be generated. 17 Collagen's primary structure is its amino acid sequence, which is a repeating triplet of G-P-X (X can be Pro or HydroxyPro among others). Secondary structure is the left-handed helical conformation. Tertiary structure is essentially the same as its secondary structure as it is a fibrous repetitive protein. Quaternary structure is the association of subunits and and the arrangment of three chains in a right-handed triple helix (coiled coil); a3. Procollagen has globular domains at the N- and C-termini, which direct the coiling of the three strands. 18 Edman Degradation 19 a) SDS is a detergent would interfer with the hydrophobic interactions and perturb secondary, tertiary, and quaternary structures, but not the primary. b) If a protein had disulfide bonds, these would be broken. This might be considered disruption of primary (covalent) structure, but it might also affect tertiary and quaternary structure, maybe some secondary. c) Trypsin will catalyze the hydrolysis of peptide bonds. This will affect the primary structure. As such, all other structures may potentially be affected (secondary, tertiary, and quaternary structure). 20 Only circle "c" 21 a. there are 2 turns of the helix in the diagram, so that would be 2 x 3.6 AA/turn = 7 AA. b. "A" belongs to the stabled -helical spectrum (minima at210 & 222 nm) "B" belongs to the un-stapled peptide as a coil with minima at 200 nm. 22 Page 3 No. on Test Correct Answer The amino-terminal residue is Gly. Cyanogen bromide cleavage gave the 22 following: Gly-(Phe, Gly)-Met/Leu-(Phe, Arg, Gly, Ala, Pro). Trypsin cleaves on the carboxyl side of Arg, so after cleavage by trypsin, there is: Gly-(Phe, Gly)-Met-Leu-Arg/Phe-(Gly, Ala, Pro). Chymotrypsin hydrolyzes the amino side of Phe, which gives the N-terminal tripeptide as a glycylglycylphenylalanine tripeptide. The other expected cleavage at the second Phe is precluded by an adjacent Pro (it can't be at the C-term as the FDNB gave Phe at the N-term of the tryptic tetrapeptide). Therefore: Gly-Gly-Phe-Met-Leu-Arg-Phe-Pro-(Ala, Gly) The sequence of the carboxyl-terminal amino acids cannot be resolved without additional information from MS/MS. The tryptic tetrapeptide is either FPAG or FPGA. The MW data from the MS/MS data shows evidence for release of a terminal Gly (MW=75) rather than a terminal Ala (MW=89). Also, there is evidence of a FPA fragment (MW=369) rather than a FPG (MW=355). Therefore, the final sequence is: Gly-Gly-Phe-Met-Leu-Arg-Phe-Pro-Ala-Gly -23 1. 2. 3. 4. C (a-helix or b-sheet) E A D 1. 2. 3. 4. 5. 6. 7. 8. G A D H E C B F 24 25 1. 2. 3. 4. 5. G J D F B 26 1. 2. 3. 4. 5. 6. C E E and C B (not wrong is C or E included as second) D B (not wrong is C or E included as second)