BIO th September, 1997

Transcription

1 BIO th September, 1997 EXAM I This exam will be taken apart for grading. Please PRINT your name on each page. If you do not have sufficient room for your answer in the space provided, please continue on the back of the page on which the question appears. Question Maximum Points Earned Points I II III IV V VI VII VIII IX X NOTE: FULL CREDIT WILL BE GIVEN FOR THOSE ANSWERS WHICH CLEARLY ADDRESS ALL RELEVANT ASPECTS OF THE QUESTIONS IN THE CLEAREST AND MOST CONCISE MANNER.

2 I. [ 8 points] Bovine ribonuclease A is a single subunit enzyme that catalyzes the hydrolysis of RNA molecules.describe how the use of urea and ß-mercaptoethanol elucidated the role of the primary structure of the protein in the determination of its tertiary structure. II. [20 points] Define and discuss briefly the significance of any five of the following. [You may want to use very simple graphs or equations. Only the first five answers will be graded]. A. alpha-helix B. beta-turn C. chaperonin D. EF hand E. protein disulfide isomerase F. allosteric enzyme G. proximal histidine H. ELISA I. Western J. circular dichroism K. homoserine lactone L. V M M. K M N. R T

3 III. [14 points] The composition of a peptide secreted by a culture of bacterial cells is: Amino Acid # Residues/peptide Cys 2 Arg 1 Glu 1 Ile 1 Leu 1 Considering ALL of the information listed below, deduce the most likely structure of the peptide. For full credit, state what EACH observation tells you about the peptide, even though later data may contradict your tentative conclusions. Your final answer must be consistent with ALL of the information provided. a. The titration curve for the peptide, over the ph range 2 thru 13, shows only two, well defined inflection points. b. The peptide: 1) does not react with iodoacetate 2) does not react with phenylisothiocyanate or dabsyl chloride 3) does not change in size when treated with trypsin c. The product of tryptic digestion reacts with fluorodinitrobenzene. d. After treatment with trypsin, three rounds of Edman degradation yielded the following partial sequence: Cys-Leu-Glu (equipment malfunction prevented further analysis). e. Following the incubation with excess 2-mercaptoethanol, the peptide binds two carboxymethyl groups w f. Estimate the isoelectric point of the untreated peptide. For full credit the structure you deduce must be consistent with EACH of the stated experimental observations. Please use the single letter codes (see last page of the exam) for each of the amino acids to write your final answer. STRUCTURE OF THE UNTREATED PEPTIDE

4 IV. [ 8 points; The point assignment corresponds to the number of correct answers expected.] Match the forms of myoglobin in the left column with their corresponding properties from the right column. 1) Metmyoglobin a) Iron in the +2 oxidation state 2) Oxymyoglobin b) Iron in the +3 oxidation state 3) Deoxymyoglobin c) Oxygen bound to the sixth coordination position of the iron d) Empty sixth coordination position e} Histidine at the fifth coordination position f) ß-chains cross-linked by BPG V. [8 points] The partial pressure of oxygen in the capillaries of active muscle is approximately 20 torr, while that in the alveolar capillaries of the lungs is approximately 100 torr. The P 50 of Mb and HbA for O 2 are 1 and 26 torr, respectively. In Hb Ranier, Tyr 145 is replaced by His thus modifying the salt bridges in deoxyhb Ranier ; P 50 for this mutant Hb is 12.9 torr. Please address the following issues; in each instance, your rationale must be clear. A. Sketch the saturation curves for Mb, HbA, and Hb Ranier, and compare their relative affinities for O 2. Please label clearly. B. Would you expect the indicated salt bridge in deoxyhb Ranier to be stronger or weaker than in deoxyhba? Why? C. Explain the fact that, as a means of compensating for the mutation, individuals with Hb Ranier, exhibit polycythemia [an increase in numbers of erythrocytes (red blood cells) and in hemoglobin].

5 VI. [10 points] The graph below is a plot of v vs v/[s] for an enzyme which follows Michaelis-Menten kinetics. This type of plot is one of several linearized forms which are used to analyze such data. 1) Which parameter(s) is(are) reflected by the slope of the lines? 2) What is the significance of the intercept on the v axis? 3) One line represents data obtained in the absence of an inhibitor, while the other represents the results obtained with an inhibitor. Which is which? What type of inhibitor is indicated? Explain your rationale. V A B v/[s]

6 VII. [9 points] ANSWER A OR B BUT NOT BOTH. ONLY YOUR FIRST ANSWER WILL BE GRADED. A. Illustrate your knowledge of the mode of action of bovine pancreatic trypsin inhibitor BPTI in as much detail as you are able. For full credit, among other things, your answer must include: 1) identification of the residue(s) on BPTI which are essential for binding to trypsin. 2)clear, accurate, specific statements regarding the relationship between the recognition site on trypsin which is responsible for its high affinity for BPTI and the catalytic triad of the protease. 3) a prediction of the effect on the BPTI:TRYPSIN COMPLEX of substituting a E for the residue normally located at position 189 of trypsin. Explain your rationale. 4)a prediction of the effect on the BPTI:TRYPSIN COMPLEX of substituting a K for the residue normally located at position 189 of trypsin. Explain your rationale. B. The three-dimensional structure of a hydrolytic antibody (17E8 ) bound to a transition state analog has been solved to 2.5 angstrom resolution. The rate enhancement (k cat /k uncat ) observed for 17E8 is 2.2 x 10 4 ; hydrolysis is enantioselective for amino acid esters with the S(L) configuration. The structural and kinetic data are complementary and support a hydrolytic mechanism for the antibody that is remarkably similar to that of the serine proteases, including evidence for formation of an acyl-enzyme intermediate. The antibody active site contains a Ser-His dyad similar to that of other serine proteases. [Taken from: Zhou, et al. (1994) Crystal Structure of a Catalytic Antibody with a Serine Protease Active Site. Science 265, ] 1) The k CAT for trypsin and 17E8 are ~ 3,500min -1 and 570min -1, respectively. Offer an explanation for the differences in values for k CAT., based on what is known about the mechanism of action of serine proteases. 2) On the basis of the information provided, would you expect trypsin and 17E8 to exhibit significant differences in K M as well? Explain your rationale. VIII. [7 points]

7 Dihydrofolate reductase is among the proteins that have been frequently used in studies of the mechanism of action of the bacterial chaperonin, GroEL. One approach to the question of sequence specificity for binding to GroEL involves examination of chaperonin-mediated folding of homologous proteins derived from different species. In one such study, human DHFR was found to be quantitatively recognized by GroEL, while the DHFR from E. coli, which has a virtually identical fold is not recognized at all by GroEL. 1) Do these results have anything to say about the role of chaperonins in the folding of DHFR E. coli cells? 2) Provide an explanation for the differences in interaction between GroEL and DHFR from humans and E. coli. 3) How might you test your hypothesis? IX. [6 points] The Hsp90 heat shock protein of eukaryotic cells regulates the activity of proteins involved in signal transduction and may direct intracellular folding in general. Hsp90 performs at least part of its function in a complex with a specific set of partner proteins that include members of the peptidyl prolyl isomerase family. The properties of some of these components were examined through the use of in vitro folding assays. One of these components FKBP52, a member of the immunophylin family, has three domains, one of which exhibits peptidyl prolyl isomerase activity. FKBP52 was able to suppress the thermally induced aggregation of the test protein citrate synthase. This chaperone activity of FKBP52 was not altered in the presence of concentrations of rapamycin sufficient to completely inhibit its peptidyl prolyl isomerase activity. [Taken from: Bose, S., Weikl, T., Bugl, H. and Buchner, J. (1996) Chaperone Function of Hsp90-Associated Proteins. Science 274, ] What do these observations say about the mechanism of action of FKBP52 as a chaperone?

8 X. [10 points] In order to observe the association of polypeptides with the chaperonin system in vivo, you decide to perform pulse-chase experiments, as done by the Hartl group (Cell, 1997, 90: 491), starting from spheroplasts obtained from cells incubated at normal growth temperature. Please answer the following questions related to the mode of chaperonin-mediated polypeptide folding and your experimental results. A. (2 pts) State two unique aspects of protein folding in vivo which lead to the necessity for molecular chaperones. B. (3 points) What are the major functional elements that contribute to the high efficiency of chaperonin-assisted polypeptide folding in the cell? C. (2 pts) What is the role of EDTA in the spheroplast lysis buffer and the subsequent buffers used to isolate the GroEL-bound polypeptide chains? What does EDTA prevent during experimental manipulations? D. (3 pts) Excess nonradioactive methionine was added at time zero, and the GroEL-bound polypeptide fractions from aliquots taken at time 0, 15 sec and 100 sec were run in lane A, B and C respectively. The radiolabeled polypeptides eluted from GroEL were resolved by SDS-PAGE; the results are summarized in the figure below. The molecular weight for each band is indicated. What is the role of excess nonradioactive methionine? Why are three bands missing in lane B compared to lane A? Relative to the molecular weight of each polypeptide species, what does the gel indicate about their folding? A B C 100kD 60kD 30kD 20kD 10kD 5kD time: (sec)

9 Amino Acid Three Letter One-letter Abbreviation Symbol Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic Acid Asp D Aspargine or Aspartic Acid Asx B Cysteine Cys C Glutamine Gln Q Glutamic Acid Glu E Glutamine or Glutamic Acid Glx Z Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V