Chemistry 27 Spring 2005 Exam 1 Chemistry 27 Professor Gavin MacBeath arvard University Spring 2005 our Exam 1 Friday, February 25, 2005 11:07 AM 12:00 PM Discussion Section (Day, Time): TF: Directions: 1. Do not write in red ink. othing in red ink will be graded. 2. Write your name on every page of the exam. 3. Provide your answers in the designated space. 4. The last page of the exam has a table of amino acids, cofactors, and DA bases for you to use. You may use the back of this amino acid page for scratch work but nothing written on this page will be graded. 5. You have 11 total pages. We have provided plenty of space for each answer. 6. All intermediates must be drawn as a distinct step. You need not draw out proton transfers as a distinct step unless explicitly told to do so. 7. Molecular model kits and calculators are permitted. 8. A significant fraction of your exams are photocopied before they are returned. Question Score 1 /20 2 /20 3 /20 4 /20 5 /20 Total /100 Page 1 of 12
1. [20pts] Consider molecule A shown below. Me A) Draw the two possible chair-like conformations of molecule A. (8pts) A B) Which is the lowest energy conformation? Explain your answer. (4pts) C) For molecule B shown below, draw a ewman projection of its lowest energy conformation, sighting down the central S-S bond. Discuss briefly the two opposing forces that determine this rather unusual dihedral angle. (8pts) Me S S Me B verlap of the σ * of the S-Me bond with the lone pair on the sulfur (hyperconjugation) is best in the gauche conformation. Steric interference is minimized in the staggered conformation. The 90 o angle is a compromise it allows fro hyperconjugation while minimizing steric interference. Page 2 of 12
2. [20pts] Phospholipids are molecules that contain a charged, polar group ( head ) and a long, nonpolar group ( tail ). The membrane of a cell consists of a phospholipid bilayer in which the charged, polar head groups are directed outward (toward the aqueous environment) and the nonpolar tails form the inside of the bilayer. In lecture, we focused on soluble proteins that are found in aqueous environments, either inside or outside cells. Some proteins, however, are found embedded in the cell membrane, as pictured below. A) What do you think is the driving force that causes individual phospholipid molecules to come together to form this bilayer structure (1 sentence)? With respect to the spontaneous formation of a bilayer from individual phospholipids in aqueous solution, please indicate the sign of and S (circle the correct answer). (4pts) As in protein folding, the release of the ice-like water upon formation of the bilayer makes the process energetically favorable. is positive or is negative S is positive or S is negative 2pts for sentence, 1pt each for and S Page 3 of 12
B) Proteins that are embedded in the cell membrane fold within that nonpolar environment to adopt a specific three-dimensional structure. Based on your understanding of thermodynamics, please indicate whether or not each of the following contributes significantly to the overall free energy of folding ( G) of a membrane protein. If the contribution is significant, please indicate whether the contribution is primarily enthalpic ( ) or entropic ( S). i). ydrogen Bonding (3pts) (circle the appropriate answer) Contributes significantly or Does not contribute significantly to G of folding. Contribution is largely enthalpic or Contribution is largely entropic Unfolded form no -bonding with environment; folded form extensive -bonding within the protein. Folding of protein involves the net formation of -bonds. contribution is significant and largely enthalpic. ii). Electrostatic Interactions (3pts) (circle the appropriate answer) Contributes significantly or Does not contribute significantly to G of folding. Contribution is largely enthalpic or Contribution is largely entropic Unfolded form no electrostatic interaction or solvation with environment; folded form extensive electrostatic interactions within the protein. Folding of protein involves the net formation of electrostatic interactions. Contribution is significant and largely enthalpic. iii). Disulfide Bonds (3pts) (circle the appropriate answer) Contributes significantly or Does not contribute significantly to G of folding. Contribution is largely enthalpic or Contribution is largely entropic Disulfides reduce the entropy of the unfolded protein but have little effect on the entropy of the folded protein. Contribution is significant and largely entropic. iv). ydrophobic Effect (3pts) (circle the appropriate answer) Contributes significantly or Does not contribute significantly to G of folding. Contribution is largely enthalpic or Contribution is largely entropic Water is largely absent and so, therefore, is the hydrophobic effect. Does not contribute significantly. 2pts for significance, one point for enthalpic/entropic Page 4 of 12
C) You have just synthesized an artificial protein that resides entirely in the membrane and folds to adopt a single, stable structure. You measure and S of folding at room temperature. Based on your understanding of thermodynamics, please circle the most likely result: is positive or is negative (2pts) S is positive or S is negative (2pts) I have no understanding of thermodynamics (0pts) Since the most significant contribution to free energy of folding comes from -bonds and electrostatic interactions, is negative. Since the entropy of the environment does not change significantly, and since the entropy of the protein decreases when it folds, S is negative. o partial credit Page 5 of 12
3. [20pts] The hormone GLP-1 is a small protein that plays a key role in glucose homeostasis and is a significant target for diabetes treatment. Unfortunately, the human version of the protein has a half-life of 90 seconds, far too short to make an effective drug candidate. Recently, investigators isolated Exendin-4, a 39 amino acid protein from the saliva of the Gila Monster, which binds to GLP-1 receptors and significantly increases insulin production. This protein is stable for 2-4 hours in the human body. In order to study Exendin-4, you must determine its primary sequence. It is too long to sequence by Edman degradation, so you first digest the protein with cyanogen bromide, which produces two fragments Frag I and Frag II. A) Frag I is short enough to be sequenced by Edman degradation and has the following sequence: AGPGTFTSDLSKQM. Draw the mechanism for the first round of Edman degradation up to the thiozolinone derivative (the kinetic product). You may abbreviate the unaffected portion of the peptide as R. (10pts) Page 6 of 12
B) Draw the final products (after treatment with strong acid) of the first three rounds of Edman degradation. (6pts) C) Frag II, the larger fragment, must be further digested before sequencing. When digested with chymotrypsin which cleaves after F, Y, and W, the products are: (i) LKGGPSY (ii) EEEAVRLF (iii) GAKPPS (iv) IEW When digested with trypsin which cleaves after R and K, the products are: (i) GGPSYGAK (ii) LFIEWLK (iii) EEEAVR (iv) PPS What is the sequence of Frag II? (4pts) + - EEEAVRLFIEWLKGGPSYGAKPPS C - o partial credit Page 7 of 12
4. [20pts] An unusual protein was isolated from a thermophilic bacterium which seems to contain several non-natural amino acids. You decide to investigate this protein by digesting it with trypsin and running it on a tandem mass spectrometer. The idealized spectrum, showing only the b-ions, is provided below. Counts b1 = 115.0 b2 = 271.1 b3 = 457.2 R W C Ω Q T G b4 = 560.2 b5 = 677.2 b6 = 805.3 b7 = 906.3 b8 = 963.3 100 200 300 400 500 600 700 800 900 1000 m/z (Da) A) For most peptides, the b 1 ion is not observed because it is not able to be stabilized in the same way as all other b-ions. When the first amino acid is Asn, however, the b 1 ion is more easily observed. Please provide two alternative mechanisms showing the rearrangement of the Asn b 1 ion, both of which lead to a more stable ion. (10pts) Page 8 of 12
B) The non-natural amino acid Ω is also observed in this spectrum. What is the molecular weight of Ω in solution at p 7? (5pts) C) Further chemical studies of Ω show the presence of a sulfur however, this amino acid is not capable of forming a disulfide bond. Suggest a possible structure for Ω? (5pts) Atom MW 14.0 C 12.0 16.0 1.0 S 32.0 Page 9 of 12
5. [20 pts] You wish to make cyclo-gavi, I, a cyclic peptide. A native chemical ligation would be a clever way to achieve cyclization; unfortunately, cyclo-gavi has no cysteines. A new method, utilizing a modified amino acid (containing an α -auxillary group), has been developed, however, allowing for native chemical ligations in the absence of cysteines. AVI I A) Starting with DCC-activated, auxillary-modified Gly and the rest of the peptide (AVI) already protected and attached to a solid-support, draw an arrow pushing mechanism for the incorporation of the -terminal Gly. You may use the structure provided below for the first arrow-pushing step. (8pts) Boc Cy Cy 2 AVI S S MeBzl Page 10 of 12
B) At this point, you treat the -terminal modified peptide with F, followed by benzyl mercaptan, affording peptide II. When II is placed in water at p 7.4, it spontaneously cyclizes. Draw an arrow-pushing mechanism for the cyclization of II to yield cyclic peptide III. (12pts) AVI S AVI S 2 p 7.4 S II III Page 11 of 12
Useful Information Me S Alanine Ala/A Cysteine Cys/C Aspartate Asp/D Glutamate Glu/E Phenylalanine Phe/F 3 Glycine Gly/G istidine is/ Isoleucine Ile/I Lysine Lys/K Leucine Leu/L 2 2 S Me 2 2 2 Methionine Met/M Asparagine Asn/ Proline Pro/P Glutamine Gln/Q Arginine Arg/R 3 C 3 C C 3 Serine Ser/S Threonine Thr/T Valine Val/V Tryptophan Trp/W Tyrosine Tyr/Y Page 12 of 12