BI/CH421)Biochemistry)I) )Name:) ) Exam%2))))))))))10/20/2014)

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BI/CH421BiochemistryIExam210/20/2014Name:Last Name (PRINT):First hoiceMultiplechoiceMultiplechoiceTrue false andFill in theBlankProtein foldingand ansylchloride1011beta pledpeptides5Xstallographyand 2D-NMR1614158total150 pointsPage1of15Totalptsforpg

ons: READ INSTRUCTIONS BEFORE BEGINNING EXAM.1) Carefully read question before answering. Often I highlight very importantinformation so please make note when I do so to make sure you are answering thequestion 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 needadditional space, you can write on the back of the SAME page. If you do this, you mustwrite “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 readcannot receive full credit. Additionally, extremely lengthy responses containing bothcorrect and incorrect statements will be graded accordingly. Meaning, if you answer thequestion correctly but if you go on to write a “kitchen sink” response containing incorrectinformation, 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 acceptedfor a regrade request6) No use of calculators will be permitted on the exam, no 0R 8.314J K- ‐1 mol- ‐1 1.987cal K- ‐1 mol- ‐1Page2of15Totalptsforpg

BI/CH421BiochemistryIExam210/20/2014Name:Part I: Multiple choice –Circle the choice that best answers the question. Do not writethe letter in the margin to indicate your answer, circle it. 3 points each.1. Identify the most conservative amino acid substitution, assuming that these tworesidues occur at the same position in two homologous proteins.a. Leu à Valb. Leu à Proc. Leu à Aspd. Leu à Lyse. Leu à Trp2. The ramachandran plot for prolinea. 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 conformatione. would be identical to those of the other amino acids because it is also anL-amino acid3. You must cleave the following peptide into smaller fragments,NMTQRGCKTVNTFVAEPLKDVQNVCFLEWhich of the following statements about cleaving this peptide are true?a. Cleavage with cyanogen bromide would yield two fragments of roughlyequal sizeb. Trypsin cleavage would create 3 fragmentsc. Endopeptidase V8 (which cleaves on the C-terminal side of glutamateresidues) would create two polypeptides and a single glutamate residued. Elastase treatment would create two fragmentse. none of the above4. For the two conformations A and B shown below, which of the following statementsabout proline’s peptide bond is true?ABRa.b.c.d.e.RA and B are both in the cis conformationA and B are both in the trans conformationneither A or B show a proline peptide bondA is in the cis conformation and B is in the trans conformationA is in the trans conformation and B is in the cis conformationPage3of15Totalptsforpg

BI/CH421BiochemistryIExam210/20/2014Name:5. You purified a 20 kDa protein and determined its mass spectrum and got the datashown below. Which of the following statements about the data are true? (thenumbers above each peak are the m/z for that peak)a. you must have more than one protein in the sample because there is morethan one peak in the mass spectrumb. the peaks with a higher m/z are more charged than those with the lowerm/zc. the peak at m/z 1001.0 has a z 20d. 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 keyinformation not given in this question.6. For the portion of a peptide shown below, which of the following statements is trueBOCH3HNNHOACa.b.c.d.e.arrow A is pointing to the phi bond, B is pointing to the psi bondarrow B is pointing to the phi bond, C is pointing to the psi bondarrow C is pointing to the phi bond, A is pointing to the psi bondarrow C is pointing to the phi bond, B is pointing to the psi bondarrow A is pointing to the phi bond, C is pointing to the psi bond7. Which of the following amino acid pairs would exhibit identical electron densities in a2.0 Å crystal?a. Asp and Glub. Asp and Valc. Asp and Glnd. A and Ce. none of the abovePage4of15Totalptsforpg

BI/CH421BiochemistryIExam210/20/2014Name: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 formatione) Peptide bonds are also known as amide bonds.9. Which level of protein structure is defined as "the three dimensional arrangement ofpolypeptides in a protein comprised of multiple polypeptides"?a) Primaryb) Secondaryc) Tertiaryd) Quaternarye) none of the above10. Which one of the following statements about α helices is FALSE?a. The α helix is a right handed helixb. Each residue in the α helix creates to a 100 turn of the helixc. An α helix has an overall macrodipole, where the C-terminus is partiallypositively charged and the N-terminus is partially negatively chargedd. The core of the helix is tightly packed and consists of main chain atomse. The hydrogen bonds that hold the helix together parallel to the axis of thehelix.11. If the polypeptide chain shown below were in an alpha helix, then the alpha aminogroup of amino acid 5 (the one with the R5 side chain) would be in a hydrogen bondwith the C O . 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 above12. Proteins that facilitate folding of proteins into their native state inside the cell arecalleda. foldasesb. endopeptidasesc. chaperonesd. prionse. amyloidPage5of15Totalptsforpg

BI/CH421BiochemistryIExam210/20/2014Name:13. You cleave a peptide with chymotrypsin and sequence the resulting peptides andget the following :1. Met-Arg-Ala-Tyr2. Asp-Met-Leu-Phe3. Gly-AsnThe C-terminal amino acid of the starting peptide (the one cleaved withchymotrypsin) isa. Tyrb. Phec. Asnd. all the abovee. not enough information is given to answer this question14. You cleave the SAME PEPTIDE as the one digested with chymotrypsin above withcyanogen bromide. Following sequencing, you get the resulting peptides1. Arg-Ala-Tyr-Gly-Asn2. Leu-Phe-Met3. Asp-MetUsing the info in this question and #13 above, the N-terminal amino acid of theoriginal peptide is :a. Argb. Leuc. Aspd. Mete. Asn15. Which of the following polypeptides is most and least likely to form an alpha helix?Peptide 1: SEDNFGAPKSILWPeptide 2: QKASVEMAVRNSGa. peptide 1 will most likely form a helix, peptide 2 least likely to form a helixb. peptide 2 will most likely form a helix, peptide 1 least likely to form a helixc. both peptides have roughly the same likelihood to form a helixd. neither peptide would likely form a helixe. you cannot predict secondary structure from primary structure16. In X-ray crystallography, the resolution of a structurea. must be at atomic resolution (below 1.2 Å) to begin to see hydrogen atomsb. is said to be very low if it is above 6 Åc. is usually in the range of 1.5 – 3 Å for typical crystal structuresd. all of the abovee. A and C abovePage6of15Totalptsforpg

BI/CH421BiochemistryIExam210/20/2014Name:17. Shown below is a portion of a multiple sequence alignment.information you can say:Position1234HumanDIRAChickenDLRSFruit ed on this5QEQEEQ6YLPYDGPositions 1, 3, and 5 contain absolutely conserved residuesPositions 1 and 5 are the most conserved positions in this sequencePositions 2 and 6 are the least conserved positions in this sequenceall of the above are truenone of the above are true18. Ten microliters of a protein solution is diluted into 990 µL of a buffer solution, then50 µL of this diluted sample is added to 450 µL of buffer. The absorbance of thissolution is determined to be 0.1 in a 1 cm cuvette. If the extinction coefficient of theprotein is 1.0 (mg/mL)-1 cm-1, what was the starting concentration of the proteinsolution?a. 0.1 mg/mLb. 1.0 mg/mLc. 10 mg/mLd. 100 mg/mLe. 1 µg/mL19. Which of the statements about circular dichoism is not accurate?a. The CD signal arises from differential absorption of left-handed and righthanded circularly polarized light.b. Because peptide bonds absorb light in the far UV (190-240 nm), thisregion is particularly sensitive to protein secondary structural elementsc. Because aromatic side chains absorb light in the far UV (190-240 nm), thisregion is also particularly sensitive to protein tertiary structural elements.d. Relative to NMR and X-ray crystallography, CD is a very low resolutionmethod to obtain information about a polypeptide’s structuree. The CD spectrum of a globular protein would significantly change if theprotein 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 FMALDI is an ionization technique for mass spectrometry which involvessuspension of the peptides (or other biomolecules) in an organic matrix followed byvaporization using a laser.Page7of15Totalptsforpg

BI/CH421BiochemistryIExam210/20/2014Name:21. T FFibrous proteins are highly elongated molecules whose structures aredominated by a single type of regular secondary structural element.22. T FDisulfide bonds are most often found on intracellular proteins (as opposedto extracellular) due to the oxidizing environment found inside the cell.23. T FProtein primary structures are generally more highly conserved thanprotein tertiary structure.24. T FInside the cell, many metalloproteins (proteins requiring a metal for theirfunction) require specialized proteins called metallochaperones to help themcomplete 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 prolineside chains of collagen, as well as distantly related homologs required for otherprocesses, including hypoxia sensing and epigenetic regulation. Based on thisinformation, we can say the other human enzymes which maintain some sequenceand structural similarity to collagen prolyl hydroxylase but possess divergentfunctions are of human collagen hydroxylase.26. In typical protein NMR experiments to probe the three dimensional structures ofproteins, one can observe a through space interaction called a(n)(abbreviation OK) between two protons separated by many amino acids in theprimary structure as long as they are within Å of each other in the nativestructure.27. Short peptides ( 25 residues) can be directly sequenced through the use of amass spectrometer where the peptide collides with chemicallyinert atoms to induce fragmentation of the peptide. Using the m/z of the resultingfragments, the peptide sequence can then be reconstructed.28. In the process called , phenylisothiocyanate (below) reactswith the N-terminal amino group of a polypeptide, ultimately cleaving the peptidebond to release a phenylthiohydantoin derivative of the amino acid.Page8of15Totalptsforpg

BI/CH421BiochemistryIExam210/20/2014Name:Part V: Short answer29. Is protein folding a random or an ordered process? Briefly justify your answer byexplaining the key observation/contribution made by Levinthal that gave us insightinto this question. (5 points)30. Titration of Histidine:a. If you titrated imidazole (pKa 7.0) with a strong base and compared it to thetritration curve for histidine, what differences would you observe in the resultingcurves? Name and briefly describe one significant difference you would observe.Feel free to draw something to help illustrate your point if you would like. (4points)b. You are a laboratory teaching assistant and your student writes that at a pH ofabout 4, the ionization state of histidine that would predominate in solution wouldbe 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)OOH 2NNH 3NPage9of15Totalptsforpg

BI/CH421BiochemistryIExam210/20/2014Name:31. You are new lab technician hired to complete dansyl chloride treatments of proteinsamples.a. What is dansyl chloride (shown below) often used for in protein biochemistrylaboratories? (4 points)b. In the structure of dansyl chloride above, circle the reactive portion of themolecule (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 dissolvingthe protein sample in 50 mM bicarbonate buffer at pH 9.5-10.5. You do not knowhow to make bicarbonate buffer so you look around the lab and find someaminobutyric 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 20amino acids. What is going on? The chemical structures and relevant pKa’s forthese buffers is shown below, you might find that helpful to answer the question.(4 points)aminobutyric acid, pKa 10.5OHOOHCarbonic acid pKa 10.3Page10of15Totalptsforpg

BI/CH421BiochemistryIExam210/20/2014Name:32. Draw a two stranded antiparallel sheet below. Complete the picture by drawing asecond strand with AT LEAST 3 amino acids (you can just write “R” for the sidechains as I did for the one I drew). Draw ANY and ALL of the hydrogen bonds thatwould connect your strand to mine as dotted or dashed lines. I drew my peptide inthe middle so that if you make a mistake drawing on one side, you can cross that outand draw on the other side. 5 pointsRNHOROHNRNHPage11of15HNOTotalptsforpg

BI/CH421BiochemistryIExam210/20/2014Name: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 thediagram 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 whatregion(s) on the graph above. (2 points)c. You analyze the phi and psi angles of amino acid shown below, plot the angleson the graph above by writing “c” in the region where this amino acid’s torsionangles would be plotted.(5 points)d. If you analyzed the main chain torsion angles of soluble, folded synthetic (madein the lab by a chemist, not by a cell) proteins comprised of D-amino acids, whatmight you observe? Circle the letter above the correct ramachandran diagramfor D-amino acids below and then briefly justify your answer. 5 pointsPage12of15Totalptsforpg

BI/CH421BiochemistryIExam210/20/2014Name:34. Recently, the research group of Craig Crews at Yale University developed a methodto selectively remove a protein from the cell by “disguising” it as an unfolded proteinthat needs to be degraded by the cell (see figure below). The method works byengineering the target protein (the one they want to become sensitive to their smallmolecule; step1) to covalently interact with the molecule A shown below. Withoutmolecule 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 iscovalently modified with A (step 2 bottom). The labeled target is then recognized asan unfolded protein by the cellular machinery and subsequently brought to theproteasome, a molecular recycling center that chops the protein up into its aminoacid building blocks (step 3).Hypothesize about the molecular mechanism that drives this new method toselectively remove a protein of interest from a cell. How/Why does A send a signalthat the target is an unfolded or misfolded protein in need of “recycling” by theproteasome? (5 points)Page13of15Totalptsforpg

BI/CH421BiochemistryIExam210/20/2014Name:35. Several biotechnology/pharmaceutical companies have explored using “stapled”peptides to stabilize small peptides into alpha helical conformations. Unnaturalamino acids with reactive side chains are introduced into these peptides and theninduced to covalently crosslink to form the “molecular staple” that stabilizes thealpha helical conformation of the peptide (see onformationYa. For the amino acids X and Y to form the crosslink shown above, how far apart inthe primary structure of the peptide must they be? Briefly explain/justify youranswer. (4 points)b. Before and after stapling, the circular dichroism spectra shown below werecollected. Which spectrum A or B likely belongs to the stapled polypeptide?Briefly justify your answer. (4 points)ABPage14of15Totalptsforpg

BI/CH421BiochemistryIExam210/20/2014Name:36. In protein structure determination via X-ray crystallography, what role doesmolecular 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 thepeak. 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 nucleithrough bonds? What type(s) of information can be gained from this kind of NMRexperiment? 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)Page15of15Totalptsforpg

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

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