Biochemistry Exam 2 Assignment

Biochemistry  I  Exam  2                     Last Name (PRINT): First Name: Pg Topic 3 Multiple choice Multiple choice Multiple choice Multiple choice Multiple choice True false and Fill in the Blank Protein folding and His titration 4 5 6 7 8 9 Pts Total possible 12 9 15 12 11 18 13 10 Dansyl chloride 10 11 beta sheet 5 12 ramachandran 16 13 unfolded protein disguise stapled peptides 5 Xstallography and 2D-NMR 16 14 15 8 total 150 points                        Page            1            of    15   Total  pts  for  pg  _________   Biochemistry  I  Exam  2                     Instructions: READ INSTRUCTIONS BEFORE BEGINNING EXAM. 1) Carefully read question before answering. Often I highlight very important information so please make note when I do so to make sure you are answering the question correctly. 2) Write your FULL name above and at least your last name on every page. 3) Write all of your answers on the exam paper itself in the space provided. If you need additional space, you can write on the back of the SAME page. If you do this, you must write “ON BACK” so that we know where to look for your answer. 4) Your answers should be brief and legible. A correct answer that cannot be read cannot receive full credit. Additionally, extremely lengthy responses containing both correct and incorrect statements will be graded accordingly. Meaning, if you answer the question correctly but if you go on to write a “kitchen sink” response containing incorrect information, you will not receive full credit for that answer. 5) You must write your final answers in pen – no tests taken in pencil will be accepted for a regrade request 6) No use of calculators will be permitted on the exam, no exceptions. Number   Log   2 3 4 5 6 7 8 9 10 Ln   0.30 0.48 0.60 0.70 0.78 0.85 0.90 0.95 1 0.69 1.10 1.39 1.61 1.79 1.95 2.08 2.20 2.30 R  =  8.314  J•K-­‐1•mol-­‐1  =  1.987  cal•K-­‐1•mol-­‐1                        Page            2            of    15   Total  pts  for  pg  _________   Biochemistry  I  Exam  2                     Part I: Multiple choice –Circle the choice that best answers the question. Do not write the letter in the margin to indicate your answer, circle it. 3 points each. 1. Identify the most conservative amino acid substitution, assuming that these two residues occur at the same position in two homologous proteins. a. Leu à Val b. Leu à Pro c. Leu à Asp d. Leu à Lys e. Leu à Trp 2. The ramachandran plot for proline a. would have the all phi angles clustered around -60˚ b. would have all psi angle clustered around -60˚ c. would have all of both phi and psi angles clustered around -60˚ d. would show proline’s peptide bond is more stable in the cis conformation e. would be identical to those of the other amino acids because it is also an L-amino acid 3. You must cleave the following peptide into smaller fragments, NMTQRGCKTVNTFVAEPLKDVQNVCFLE Which of the following statements about cleaving this peptide are true? a. Cleavage with cyanogen bromide would yield two fragments of roughly equal size b. Trypsin cleavage would create 3 fragments c. Endopeptidase V8 (which cleaves on the C-terminal side of glutamate residues) would create two polypeptides and a single glutamate residue d. Elastase treatment would create two fragments e. none of the above 4. For the two conformations A and B shown below, which of the following statements about proline’s peptide bond is true? A B R a. b. c. d. e. R A and B are both in the cis conformation A and B are both in the trans conformation neither A or B show a proline peptide bond A is in the cis conformation and B is in the trans conformation A is in the trans conformation and B is in the cis conformation                        Page            3            of    15   Total  pts  for  pg  _________   Biochemistry  I  Exam  2                     5. You purified a 20 kDa protein and determined its mass spectrum and got the data shown below. Which of the following statements about the data are true? (the numbers above each peak are the m/z for that peak) a. you must have more than one protein in the sample because there is more than one peak in the mass 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 thought. e. you cannot interpret this mass spectrum without knowing some key information not given in this question. 6. For the portion of a peptide shown below, which of the following statements is true B O CH3 H N N H O A C a. b. c. d. e. arrow A is pointing to the phi bond, B is pointing to the psi bond arrow B is pointing to the phi bond, C is pointing to the psi bond arrow C is pointing to the phi bond, A is pointing to the psi bond arrow C is pointing to the phi bond, B is pointing to the psi bond arrow A is pointing to the phi bond, C is pointing to the psi bond 7. Which of the following amino acid pairs would exhibit identical electron densities in a 2.0 Å crystal? a. Asp and Glu b. Asp and Val c. Asp and Gln d. A and C e. none of the above                        Page            4            of    15   Total  pts  for  pg  _________   Biochemistry  I  Exam  2                     8. Which one of the following statements about peptide bonds is FALSE. a) Peptide bonds are planar. b) Peptide bonds have about 40% double bond character. c) Peptide bonds prefer to be in the cis conformation. d) Peptide bonds are important for secondary structure formation e) Peptide bonds are also known as amide bonds. 9. Which level of protein structure is defined as "the three dimensional arrangement of polypeptides in a protein comprised of multiple polypeptides"? a) Primary b) Secondary c) Tertiary d) Quaternary e) none of the above 10. Which one of the following statements about α helices is FALSE? a. The α helix is a right handed helix b. Each residue in the α helix creates to a 100˚ turn of the helix c. An α helix has an overall macrodipole, where the C-terminus is partially positively charged and the N-terminus is partially negatively charged d. The core of the helix is tightly packed and consists of main chain atoms e. The hydrogen bonds that hold the helix together parallel to the axis of the helix. 11. If the polypeptide chain shown below were in an alpha helix, then the alpha amino group of amino acid 5 (the one with the R5 side chain) would be in a hydrogen bond with the C=O of R1 R3 R5 R7 R9 O O O O H H H H H N N N N N N N N N N H H H H H R2 R4 R6 R8 O O O O O a. amino acid 1 (R = R1) b. amino acid 2 (R = R2) c. amino acid 8 (R = R8) d. amino acid 9 (R = R9) e. none of the above 12. Proteins that facilitate folding of proteins into their native state inside the cell are called a. foldases b. endopeptidases c. chaperones d. prions e. amyloid                        Page            5            of    15   Total  pts  for  pg  _________   Biochemistry  I  Exam  2   13. You cleave a peptide with chymotrypsin and sequence the resulting peptides and get the following : 1. Met-Arg-Ala-Tyr 2. Asp-Met-Leu-Phe 3. Gly-Asn The C-terminal amino acid of the starting peptide (the one cleaved with chymotrypsin) is a. Tyr b. Phe c. Asn d. all the above e. not enough information is given to answer this question 14. You cleave the SAME PEPTIDE as the one digested with chymotrypsin above with cyanogen bromide. Following sequencing, you get the resulting peptides 1. Arg-Ala-Tyr-Gly-Asn 2. Leu-Phe-Met 3. Asp-Met Using the info in this question and #13 above, the N-terminal amino acid of the original peptide is : a. Arg b. Leu c. Asp d. Met e. Asn 15. Which of the following polypeptides is most and least likely to form an alpha helix? Peptide 1: SEDNFGAPKSILW Peptide 2: QKASVEMAVRNSG a. peptide 1 will most likely form a helix, peptide 2 least likely to form a helix b. peptide 2 will most likely form a helix, peptide 1 least likely to form a helix c. both peptides have roughly the same likelihood to form a helix d. neither peptide would likely form a helix e. you cannot predict secondary structure from primary structure 16. In X-ray crystallography, the resolution of a structure a. must be at atomic resolution (below 1.2 Å) to begin to see hydrogen atoms b. is said to be very low if it is above 6 Å c. is usually in the range of 1.5 – 3 Å for typical crystal structures d. all of the above e. A and C above                        Page            6            of    15   Total  pts  for  pg  _________   Biochemistry  I  Exam  2                     17. Shown below is a portion of a multiple sequence alignment. information you can say: Position 1 2 3 4 Human D I R A Chicken D L R S Fruit Fly D V R E Mouse D I R A Zebrafish D L R K Worm D V R F a. b. c. d. e. Based on this 5 Q E Q E E Q 6 Y L P Y D G Positions 1, 3, and 5 contain absolutely conserved residues Positions 1 and 5 are the most conserved positions in this sequence Positions 2 and 6 are the least conserved positions in this sequence all of the above are true none of the above are true 18. Ten microliters of a protein solution is diluted into 990 µL of a buffer solution, then 50 µL of this diluted sample is added to 450 µL of buffer. The absorbance of this solution is determined to be 0.1 in a 1 cm cuvette. If the extinction coefficient of the protein is 1.0 (mg/mL)-1 cm-1, what was the starting concentration of the protein solution? a. 0.1 mg/mL b. 1.0 mg/mL c. 10 mg/mL d. 100 mg/mL e. 1 µg/mL 19. Which of the statements about circular dichoism is not accurate? a. The CD signal arises from differential absorption of left-handed and right handed circularly polarized light. b. Because peptide bonds absorb light in the far UV (190-240 nm), this region is particularly sensitive to protein secondary structural elements c. Because aromatic side chains absorb light in the far UV (190-240 nm), this region is also particularly sensitive to protein tertiary structural elements. d. Relative to NMR and X-ray crystallography, CD is a very low resolution method to obtain information about a polypeptide’s structure e. The CD spectrum of a globular protein would significantly change if the protein was treated with a chaotropic agent. Part II: True False (2 pts each) Circle T or F to indicate if the statement is true or false. 20. T F MALDI is an ionization technique for mass spectrometry which involves suspension of the peptides (or other biomolecules) in an organic matrix followed by vaporization using a laser.                        Page            7            of    15   Total  pts  for  pg  _________   Biochemistry  I  Exam  2                     21. T F Fibrous proteins are highly elongated molecules whose structures are dominated by a single type of regular secondary structural element. 22. T F Disulfide bonds are most often found on intracellular proteins (as opposed to extracellular) due to the oxidizing environment found inside the cell. 23. T F Protein primary structures are generally more highly conserved than protein tertiary structure. 24. T F Inside the cell, many metalloproteins (proteins requiring a metal for their function) require specialized proteins called metallochaperones to help them complete the folding process by delivering their required metals. Part III: Fill in the blank. For the following sentences, fill in the blank with the word(s) that best complete the sentence. (2 pt each blank) 25. In class we discussed prolyl hydroxylase, an enzyme required to hydroxylate proline side chains of collagen, as well as distantly related homologs required for other processes, including hypoxia sensing and epigenetic regulation. Based on this information, we can say the other human enzymes which maintain some sequence and structural similarity to collagen prolyl hydroxylase but possess divergent functions are ______________ of human collagen hydroxylase. 26. In typical protein NMR experiments to probe the three dimensional structures of proteins, one can observe a through space interaction called a(n) _______________ (abbreviation OK) between two protons separated by many amino acids in the primary structure as long as they are within _______ Å of each other in the native structure. 27. Short peptides (<25 residues) can be directly sequenced through the use of a ______________ mass spectrometer where the peptide collides with chemically inert atoms to induce fragmentation of the peptide. Using the m/z of the resulting fragments, the peptide sequence can then be reconstructed. 28. In the process called _________________, phenylisothiocyanate (below) reacts with the N-terminal amino group of a polypeptide, ultimately cleaving the peptide bond to release a phenylthiohydantoin derivative of the amino acid.                        Page            8            of    15   Total  pts  for  pg  _________   Biochemistry  I  Exam  2                     Part V: Short answer 29. Is protein folding a random or an ordered process? Briefly justify your answer by explaining the key observation/contribution made by Levinthal that gave us insight into this question. (5 points) 30. Titration of Histidine: a. If you titrated imidazole (pKa = 7.0) with a strong base and compared it to the tritration curve for histidine, what differences would you observe in the resulting curves? Name and briefly describe one significant difference you would observe. Feel free to draw something to help illustrate your point if you would like. (4 points) b. You are a laboratory teaching assistant and your student writes that at a pH of about 4, the ionization state of histidine that would predominate in solution would be the one drawn below. How would you grade the accuracy of this statement? Is it correct? If not, what specifically is incorrect about it? (4 points) O O H 2N NH 3 N                        Page            9            of    15   Total  pts  for  pg  _________   Biochemistry  I  Exam  2                     31. You are new lab technician hired to complete dansyl chloride treatments of protein samples. a. What is dansyl chloride (shown below) often used for in protein biochemistry laboratories? (4 points) b. In the structure of dansyl chloride above, circle the reactive portion of the molecule (the one that reacts with the protein functional group(s). (2 points) c. To do the dansyl chloride treatment, the protocol you are given calls for dissolving the protein sample in 50 mM bicarbonate buffer at pH 9.5-10.5. You do not know how to make bicarbonate buffer so you look around the lab and find some aminobutyric acid and use that to make up 50 mM aminobutyrate buffer instead. You try over and over again to do the labeling reaction but no matter what you do, you don’t see any products resulting from dansylation of any of the standard 20 amino acids. What is going on? The chemical structures and relevant pKa’s for these buffers is shown below, you might find that helpful to answer the question. (4 points) aminobutyric acid, pKa 10.5 O HO OH Carbonic acid pKa 10.3                        Page          10              of    15   Total  pts  for  pg  _________   Biochemistry  I  Exam  2                     32. Draw a two stranded antiparallel sheet below. Complete the picture by drawing a second strand with AT LEAST 3 amino acids (you can just write “R” for the side chains as I did for the one I drew). Draw ANY and ALL of the hydrogen bonds that would connect your strand to mine as dotted or dashed lines. I drew my peptide in the middle so that if you make a mistake drawing on one side, you can cross that out and draw on the other side. 5 points R N H O R O H N R N H                        Page          11              of    15   H N O Total  pts  for  pg  _________   Biochemistry  I  Exam  2                     33. Use the ramachandran diagram below to answer the following questions: a. What do the dark shaded regions of this plot represent? (4 points) b. If you analyzed a coiled coil domain of a protein primarily and plotted a dot on the diagram above for each amino acid’s main chain torsion angles, in what region(s) would you expect a majority of the torsion angles to appear? Indicate what region(s) on the graph above. (2 points) c. You analyze the phi and psi angles of amino acid shown below, plot the angles on the graph above by writing “c” in the region where this amino acid’s torsion angles would be plotted.(5 points) d. If you analyzed the main chain torsion angles of soluble, folded synthetic (made in the lab by a chemist, not by a cell) proteins comprised of D-amino acids, what might you observe? Circle the letter above the correct ramachandran diagram for D-amino acids below and then briefly justify your answer. 5 points                        Page          12              of    15   Total  pts  for  pg  _________   Biochemistry  I  Exam  2                     34. Recently, the research group of Craig Crews at Yale University developed a method to selectively remove a protein from the cell by “disguising” it as an unfolded protein that needs to be degraded by the cell (see figure below). The method works by engineering the target protein (the one they want to become sensitive to their small molecule; step1) to covalently interact with the molecule A shown below. Without molecule A, the target protein has its normal cellular lifetime (step 2 top). However, as soon as molecule A is added to the cells, their engineered protein target is covalently modified with A (step 2 bottom). The labeled target is then recognized as an unfolded protein by the cellular machinery and subsequently brought to the proteasome, a molecular recycling center that chops the protein up into its amino acid building blocks (step 3). Hypothesize about the molecular mechanism that drives this new method to selectively remove a protein of interest from a cell. How/Why does A send a signal that the target is an unfolded or misfolded protein in need of “recycling” by the proteasome? (5 points)                        Page          13              of    15   Total  pts  for  pg  _________   Biochemistry  I  Exam  2                     35. Several biotechnology/pharmaceutical companies have explored using “stapled” peptides to stabilize small peptides into alpha helical conformations. Unnatural amino acids with reactive side chains are introduced into these peptides and then induced to covalently crosslink to form the “molecular staple” that stabilizes the alpha helical conformation of the peptide (see below). X   unnatural  amino   acid  side  chains   before  the  covalent   crosslinking  to   create  the  “staple”   X   Y   the  crosslinked   (stapled)  peptide  is   stabilized  on  the   alpha  helical   conformation   Y   a. For the amino acids X and Y to form the crosslink shown above, how far apart in the primary structure of the peptide must they be? Briefly explain/justify your answer. (4 points) b. Before and after stapling, the circular dichroism spectra shown below were collected. Which spectrum A or B likely belongs to the stapled polypeptide? Briefly justify your answer. (4 points) A   B                          Page          14              of    15   Total  pts  for  pg  _________   Biochemistry  I  Exam  2                     36. In protein structure determination via X-ray crystallography, what role does molecular modeling play in this process? ( 5 points) 37. NMR is often used to interrogate protein structure. a. In 1D NMR experiments the signal intensity is represented by the height of the peak. How is the intensity of a signal represented in 2D-NMR data? ( 3 points) b. What 2D-NMR experiment used to examine coupling of NMR active nuclei through bonds? What type(s) of information can be gained from this kind of NMR experiment? How is that information is used to determine the protein structure? i. the name of the experiment (abbreviations OK)______________________________ (2 points) ii. the information gained from the experiment named in (i) (3 points) iii. how the information listed in (ii) is used to determine a protein structure. (3 points)                        Page          15              of    15   Total  pts  for  pg  _________