Biochemistry Exam 2 Answer Key Assignment

Biochemistry Exam 2 Part 1: Multiple choice -Circle the choice that best answers the question. Do not write the letter in the margin to indicate уоuг answer, circle it. 3 points each. 1. ldentify the most conservative amino acid substitution, assuming that these two residues оссuг at the same position in two homologous proteins. @ Leu ➔ Val Ь. Leu ➔ Рго с. Leu ➔ Asp d. Leu ➔ Lys е. Leu ➔ Тгр 2. The ramachandran plot fог proline would have the all phi angles clustered around -во· Ь. would have all psi angle clustered around -во· с. would have all of both phi and psi angles clustered around -во· d. would show proline's peptide bond is тоге staЫe in the cis conformation е. would Ье identical to those of the other amino acids because it is also ап L-amino acid ® 3. м,1Тоф 'f е tide into sma lег fragments, NTF +L�D oN CFLE Which of the following atem nts Ь ut clea ing t is peptide аге true? а. Cleavage with cyanogen bromide would yield two fragments of roughly �ze Ь. -cleava�ould сгеаtе 3 fragments с. Endopeptidase-(which cleaves оп the C-terminal side of glutamate residues would сгеаtе two polypeptides and а single glutamate residue d. Elastase reatment would сгеаtе two fragments попе of the above ® 4. Fог the two conformations А and В shown below, which of the following statements about proline's peptide bond is true? в а. Ь. с. d. � А and В аге both in the cis conformation А and В аге both in the trans conformation neither А ог В show а proline peptide bond А is in the cis conformation and В is in the trans conformation А is in the trans conformation and В is in the cis conformation Вiochemistry I Exam 2 5. You purified а 20 kDa protein and determined its mass spectrum and got the data shown below. Which of the following statements about the data аге true? (the numbers above each eak аге the m/z fог that peak) 1001.0 н)СI 1053.6 953.4 910.1 1112.1 rn/L а. you must have mоге than опе protein in the sample because there is mоге than опе peak in the mass spectrum Ь. the peaks with а higher m/z аге mоге charged than those with the lower m/z � the peak at m/z =1001.0 has а z=20 d. the protein has а mass of 1001.0 Da, not 20 kDa as you had thought. е. you cannot interpret this mass spectrum without knowing some key information not given in this question. 6. For the portion of а peptide shown below, which of the following statements is true в о\ С Нз н N i�h / А С а. аггоw А is pointing to the phi bond, В is pointing to the psi bond аггоw В is pointing to the phi bond, С is pointing to the psi bond с. аггоw С is pointing to the phi bond, А is pointing to the psi bond d. аггоw С is pointing to the phi bond, В is pointing to the psi bond е. аггоw А is pointing to the phi bond, С is pointing to the psi bond ® 7. Which of the following amino acid pairs would exhiЬit identical electron densities in а 2.0 А crystal? а. Asp and Glu (§)Asp and Val с. Asp and Gln d. A and С е. попе of the above Вiochemistry I Exam 2 8. Which опе of the following statements about peptide bonds is FALSE. а) Peptide bonds аге рlапаг. Ь) Peptide bonds have about 40% douЫe bond character. @Peptide bonds ргеfег to Ье in the cis conformation. d) Peptide bonds аге important fог secondary structure formation е) Peptide bonds аге also known as amide bonds. 9. Which level of protein structure is defined as ''the three dimensional arrangement of polypeptides in а protein comprised of multiple polypeptides"? а) Primary Ь) Secondary Tertiary Quaternary попе of the above 10. Which опе of the following statements about а helices is FALSE? а. The а helix is а right handed helix Ь. Each residue in the а helix creates to а 1 оо· turn of the helix � Ап а helix has ап overall macrodipole, where the C-terminus is partially positively charged and the N-terminus is partially negatively charged d. The саге of the helix is tightly packed and consists of main chain atoms е. The hydrogen bonds that hold the helix together parallel to the axis of the helix. U 11. lf the polypeptide chain shown below wеге in ап alpha helix, then the alpha amino group of amino acid 5 (the опе with the Rs side chain) would Ье in а hydrogen bond with the С=О of О О О О R5 R7 R3 R1 R9 Н Н Н Н Н N N N N N /N� ylN� ylN� ylN� ylN� / Н Н Н Н Н О О R О R О R О R 2 � amino acid 1 (R = R1 ) amino acid 2 (R = R2) с. amino acid 8 (R = Ra) d. amino acid 9 (R = Rg) е. попе of the above У 4 6 8 12. Proteins that facilitate folding of proteins into their native state inside the cell аге called а. foldases Ь. endopeptidases с. chaperones d. prions е. amyloid Biochemistry I Exam 2 13. You cleave а 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 опе cleaved with chymotrypsin) is а. Туг Ь. Phe Asn d. all the above е. not enough information is given to answer this question @ 14. You cleave the SAME PEPTIDE as the опе 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 а. Агg Ь. Leu (c.)Asp У.меt е. Asn 15. Which of the following polypeptides is most and least likely to form ап alpha helix? Peptide 1: SEDNFGAPKSILW Peptide 2: QКASVEMAVRNSG а. peptide 1 will most likely form а helix, peptide 2 least likely to form а helix � peptide 2 will most likely form а helix, peptide 1 least likely to form а helix both peptides have roughly the same likelihood to form а helix d. neither peptide would likely form а helix е. you cannot predict secondary structure from primary structure У 16. ln Х-гау crystallography, the resolution of а structure а. must Ье at atomic resolution (below 1.2 А) to begin to see hydrogen atoms Ь. is said to Ье vегу low if it is above 6 А с. is usually in the гапgе of 1.5 - 3 А fог typical crystal structures @ all of the above е. А and С above Вiochemistry I Exam2 17. Shown below is а portion of а multiple sequence alignment. information you сап say: 1 2 Position 3 4 Human D I R А D R Chicken L s Е D Fruit Fly R V D R Mouse I А D Zebrafish L R к D Worm V R F Based оп this 5 Q Е Q Е Е Q 6 у L р у D G Positions 1, 3, � contain absolutely conserved residues Positions 1 and �аге the most conserved positions in this sequence Positions �and 6 аге the Jeast conserved pos itions in this sequence all of the above аге true @ попе of the above аге true а. Ь. с. d. 18. Теп microliters of а protein solution is diluted into 990 µL of а buffer solution, then 50 µL of this diluted sample is added to 450 µL of buffer. The absorbance of this solution is determined to Ье 0.1 in а 1 cm cuvette. lf the extinction coefficient of the protein is 1.0 (mg/ml)- 1 cm- 1, what was the starting concentration of the protein solution? а. 0.1 mg/ml Ь. 1.0 mg/ml с. 10 mg/ml @ 100 mg/ml е. 1 µg/ml 19. Which of the statements about circular dichoism is not accurate? а. The CD signal arises from differential absorption of left-handed and right handed circularly polarized light. Ь. Because peptide bonds absorb light in the fаг UV (190-240 nm), this region is particularly sensitive to protein secondary structural elements @ Because aromatic side chains аЬsогЬ light in the fаг UV (190-240 nm), this region is also particularly sensitive to protein tertiary structural elements. d. Relative to NMR and Х-гау crystallography, CD is а vегу low resolution method to oblain information about а polypeptide's structure е. The CD spectrum of а globular protein would significantly change if the protein was treated with а chaotropic agent. Part 11: True False (2 pts each) Circle Т ог F to indicate if the statement is true ог false. 200 F MALDI is ап ionization technique fог mass spectrometry which involves suspension of the peptides (ог other Ьiomolecules) in ап organic matrix followed Ьу vaporization using а laser. 21 (!) F Fibrous proteins аге highly elongated molecules whose structures аге dominated Ьу а single type of regular secondary structural element. 22. Т fi=\ Disulfide bonds аге most often found оп intracellular proteins (as opposed to eblcellular) due to the oxidizing environment found inside the cell. 23. Т (F\ Protein primary structures аге generally more highly conserved than protbltertiary structure. 24. Т F lnside the cell, many metalloproteins (proteins requiring а metal for their function) require specialized proteins called metallochaperones to help them complete the folding process Ьу delivering their required metals. Part 111: Fill in the Ыапk. For the following sentences, fill in the Ьlank with the word(s) that best complete the sentence. (2 pt each Ыank) 25. ln 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 оп this information, we сап say the other human enzymes which maintain some sequence and structural similarity to collagen prolyl hydroxylase but possess divergent functions аге _______ of human collagen hydroxylase. 26. ln typical protein NMR experiments to ргоЬе the three dimensional structures of proteins, one сап observe а through space interaction called a(n) NOSEY NOSEY (abbreviation ОК) between two protons separated Ьу many amino acids in the primary structure as long as they аге within 5 А of each other in the native 5 structure. 27. Short peptides (<25 residues) сап Ье directly sequenced through the use of а mass spectrometer where the peptide collides with chemically tandem tandem inert atoms to induce fragmentation of the peptide. Using the m/z of the resulting fragments, the peptide sequence сап then Ье reconstructed. egradatioo , phenylisothiocyanate (below) reacts 28. ln the process called Fdman Edman d degradation g with the N-terminal amino roup of а polypeptide, ultimately cleaving the peptide bond to release а phenylthiohydantoin derivative of the amino acid. Part V: Short answer 29. ls protein folding а random ог ап ordered process? Briefly justify your answer Ьу explaining the key observation/contribution made Ьу Levinthal that gave us insight into this question. (5 points) 30. Titration of Histidine: а. lf you titrated imidazole (рКа = 7.0) with а strong base and compared it to the tritration curve for histidine, what differences would you observe in the resulting curves? Name and briefly describe опе significant difference you would observe. Feel free to draw something to help illustrate your point if you would like. (4 points) по buffering 2.3 or ог 9.6. no buffering at at 2.3 9.6. Ь. You аге а laboratory teaching assistant and your student writes that at а рН of about 4, the ionization state of histidine that would predominate in solution would Ье the опе drawn below. How would you grade the accuracy of this statement? 1s it соггесt? lf not, what specifical/y is incorrect about it? (4 points) -0 :-Nн, г!N H2N--.!/ The proton proton is is on the wrong imidazole nitrogen nitrogen (along the + + charge) charge) The оп the wrong imidazole (along with with the 31. You аге new lab technician hired to complete dansyl chloride treatments of protein samples. а. What is dansyl chloride (shown below) often used fог in protein Ьiochemistry laboratories? (4 points) N-terminal analysis N-terminal analysis ,..... N 'сн3 нс 3 Ь. ln the structure of dansyl chloride above, circle the reactive portion of the molecule (the опе that reacts with the protein functional group(s). (2 points) с. То do the dansyl chloride treatment, the protocol you аге given calls fог dissolving the protein sample in 50 mM Ьicarbonate buffer at рН 9.5-10.5. You do not know how to make ЬiсагЬопаtе 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 оvег and оvег again to do the labeling reaction but по matter what you do, you don't see апу products resulting from dansylation of апу of the standard 20 amino acids. What is going оп? The chemical structures and relevant pKa's fог these buffers is shown below, you might find that helpful to answer the question. (4 points) о H2N� он aminobutyric acid, рКа 10.5 о Jl HO OH СагЬопiс acid рКа 10.3 Aminobutyrate will will Ье be at at pH 8-9, but but with with NO The Aminobutyrate рН 8-9, NO buffering. buffering. The reaction releases will quickly reaction releases protons protons and and the the reaction reaction will quickly Ье be acidic acidic so по amiпo so that that no amino groups groups are are deprotoпated, deprotonated, which which is is needed needed for for reactioп reaction (similar (similar to to edman edman degradation) degradation) 32. Draw а two stranded antiparallel sheet below. Complete the picture Ьу drawing а second strand with АТ LEAST 3 amino acids (you сап just write "R" for the side chains as I did for the опе I drew). Draw ANY and ALL of the hydrogen bonds that would connect your strand to mine as dotted ог dashed lines. 1 drew my peptide in the middle so that if you make а mistake drawing оп опе side, you сап cross that out and draw оп the other side. 5 points 1-1)\1'�:\ С=О C-NH-C=O C-NH-C=O C=O C-NH-C=O C-NH-C=O C-NH-C=O C-NH-C=O R R R R R R 33. Use the ramachandran diagram below to answer the following questions: +180 -----. 1. What do the dark shaded regions of this plot represent? (4 points} 120 C .,.. .. 60 Favored angles Favored angles о -э. -60 -120 -180 -180 о +180 ф (degrees) !2.:_ lf you analyzed а coiled coil domain of а protein primarily and plotted а dot оп the diagram above for each amino acid's main chain torsion angles, in what region(s) would you expect а majority of the torsion angles to арреаг? lndicate what region(s) оп the graph above. (2 points) с. You analyze the phi and psi angles of amino acid shown below, plot the angles оп the graph above Ьу writing "с" in the region where this amino acid's torsion angles would Ье plotted.(5 points) О R Н Y'N�Nj li ;\ С'2 <:XN2 d. lf you analyzed the main chain torsion angles of soluЫe, folded synthetic (made in the lab Ьу а chemist, not Ьу а 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 (А) (В) +18Оrг--- ====, +180 120 -;; t :;. ( 60 о -60 ..... -120 120 � : :!! -$- 60 J � о :!!. -$- -60 -120 -180 .___ _ ___.__ ___, -180 +180 ф(devr=I е) (С)+180 120 J 60 о :о � -60 -120 о ф(devree>! +180 -180 -180 180 120 60 о -60 -120 о ф!dogree,) +180 J -180 -180 +180 ф(devree,) 34. Recently, the research group of Craig Crews at Yale University developed а method to selectively remove а protein from the cell Ьу "disguising" it as ап unfolded protein that needs to Ье degraded Ьу the cell (see figure below). The method works Ьу engineering the target protein (the опе they want to become sensitive to their small molecule; step1) to covalently interact with the molecule А shown below. Without molecule А, the target protein has its normal cellular lifetime (step 2 top). However, as soon as molecule А is added to the cells, their engineered protein target is covalently modified with А (step 2 bottom). The labeled target is then recognized as ап unfolded protein Ьу the cellular machinery and subsequently brought to the proteasome, а molecular recycling сепtег that chops the protein up into its amino acid building Ыocks (step 3). target protein target protein (51:ер 1) ► target protein target has norma/ cel/ular /ifetime -;.::::::,d target with engineered Ыnding site for "R" target into cells (step 2) (step 3) target protein target is covalently attached to the sma/1 то/еси/е /аЬе/ ► target is degraded Ьу rhe proteosome molecule А. Hypothesize about the molecular mechanism that drives this new method to selectively remove а protein of interest from а cell. How/Why does А send а signal that the target is ап unfolded ог misfolded protein in need of "recycling" Ьу the proteasome? (5 points) 35. Several Ьiotechnology/pharmaceutical companies have explo red using "stapled " peptides t o staЬilize small peptides into alpha helical conformations. Unnatural amino acid s with reactive side chains аге in troduced into these peptides and then induced to co valently crosslink t o form the "molecular staple" that staЬilizes the alpha helical c onformation of the peptide (see below). the crosslinked ( stapled) peptide is staЬilized on the alpha helical conformation х unnatural amino acid side chains before the covalent crosslinking to create the "staple" у у а. Fог the amino acid s Х and У to fo rm the crosslink show n abo ve, how fаг apart in the primary structure of the peptide m ust they Ье? Briefly explain/justify уоuг answer. (4 points) There 3.6 AA/tur n. So, So, 2х3.6=7.2, ог There аге are 2 2 turns turns o off the the helix, helix, which which is is 3.6 AA/turn. 2x3.6=7.2, or 7-8 Ад AA apart. apart. 7-8 Ь. Веfоге and after stapling, the circular dichroism spectra shown below wеге co llected . Which spectrum А ог В likely belongs t o the stapled polypeptide? Briefly justify уоuг an swer. (4 points) 80 70 60 .... • 50 А stapled stapled (alpha-helix (alpha-helix spectrum) spectrum) 40 30 8 • / ф :� •• _ 101 -20 -3 0 -40 unstapled (random coli) ndom coli) " � :� � :� : � . . .- о •••. 2о�э )���•··••2ю·"•'1з�..•••1�о , ••• ••• •1• • . .. ••••• •••••••••••••••• Wavelength (nm) 2so 260 36. ln protein structure determination via Х-гау crystallography, what role does molecular modeling play in this process? ( 5 points) Unless this is is the the part where the the molecule molecule is is fit fit into into the the density. density. We We never never defined defined this this Unless this part where explicitly explicitly as as "molecular "molecular modeling" modeling" 37. NMR is often used to interrogate protein structure. а. ln 1 D NMR experiments the signal intensity is represented Ьу the height of the peak. How is the intensity of а signal represented in 2D-NMR data? ( 3 points) Ь. What 2D-NMR experiment used to examine coupling of NMR active nuclei through bonds? What type(s) of information сап Ье gained from this kind of NMR experiment? How is that information is used to determine the protein structure? COSEY_________ i. the пате of the experiment (abbreviations OK)__c_o_s_E_Y (2 points) ii. the information gained from the experiment named in (i) (3 points) Angles, particularly the psi angles angles Angles, particularly the psi iii. how the information listed in (ii) is used to determine а protein structure. (3 points) ifif you you know psi and and phi phi angles angles you protein folds. know the the psi you know know how how the the protein folds.