Biochemistry Exam: Amino Acids, Proteins, and Interactions, Exams of Biochemistry

Download Biochemistry Exam: Amino Acids, Proteins, and Interactions and more Exams Biochemistry in PDF only on Docsity! Name ___ANSWERS_____________ Page 1 of 10 amino acid pKa Asp 3.9 Glu 4.3 His 6.0 Cys 8.3 Tyr 10.1 Lys 10.5 Arg 12.5 Ser 13.0 Thr 13.0 N-term. amino group 9.5 C-term. carboxy group 2.0 CHEM 641 Biochemistry First Exam Saturday, October 6, 2007, 10am - noon, 100 Kirkbride Instructor: Bahnson Please put your name on the front, initial each page, and check to make sure that your exam copy contains all 10 pages. During the exam, take care not to expose your answers to your neighbors. If you have any questions, raise your hand and I will come to you. Remember, no calculators please. Good luck! Breakdown of Exam Part I 10 Short Answer 10 pts Part II 5 multiple choice 10 pts Part III 2 sections 20 pts Part IV 5 questions 60 pts Total 100 pts Here are some useful constants and equations: Gas constant - R = 8.315 J mol-1 K-1 or R = 1.987 cal mol-1 K-1 ºC + 273.15 = ºK 1 cal = 4.184 Joule 1 mM = 10-3 M, 1 Å = 10-10 m Van der Waals E = B/r12 - A/r6 Ionic interaction F = e1e2/Dr2 +H3N CO2- H R L-amino acid Name ___ANSWERS_____________ Page 2 of 10 PART I. Really short Answers (10 pts) 1. Technique best suited to show polydispersity of a protein sample. _____Native PAGE_______ 2. Method to obtain crystallographic phase from a homologous protein structure. _Molecular replacement (MR)_ 3. Write the equation relating Gibbs free energy and entropy. ___∆G = ∆H - T∆S____ 4. Most significant weak force interaction of the dimerization of a protein. __hydrophobic interactions__ 5. Protein technique used to analyze purity based on antibody recognition. __Western or Immunoblot__ 6. Common motif of anti-parallel α-helix proteins. _helix-turn-helix, leucine zipper_ 7. and 8. Name one Nobel prize winner mentioned in class others were accepted also and what they did. __Perutz and Kendrew__ ____Mb and Hb structure_____ 9. The tortion angle that rotates the Cα-C bond of a protein backbone. _______PSI______________ 10. Name of through space interactions used to provide NMR distance restraints for a protein structure. _______NOE___________ Name ___ANSWERS_____________ Page 5 of 10 PART IV. - Slightly Longer Answer Problems 1. α-helices a) [5 pts] Draw how two peptide planes align in an α-helix showing the H-bond formed. Based on your drawing, predict the overall dipole of this α-helix. Explain briefly. Peptide planes are all lined up with their carbonyl oxygens pointing to the C-terminus of the helix. This leads to a dipole with a positively charged N-term of +0.5 and a C-term of –0.5. Part b [5 pts] Describe two separate reasons why the amino acid proline is not typically found in the middle of an α-helix. 1) The backbone N lacks an amide hydrogen, so it can not make a H-bond in middle of helix. 2) Due to covalent attachment of side chain to N, the Phi tortion angle is restricted to a non-helical angle, thereby giving the helix a kink if Pro is in it. Name ___ANSWERS_____________ Page 6 of 10 +H3N N N N O- O H O O H O SH NH2 O N H 2. Peptides a. [7 pts] Draw the structure of the tetra-peptide Asn-Trp-Pro-Cys as it would appear at pH 7.0 including the full structures of the side-chains. Also show proper stereochemistry around any chiral atoms of the L-amino acids. b. [3 pts] Estimate the net charge on the peptide at pH 4.3, 8.3 and 10.9. pH 4.3 __0___ pH 8.3 __-0.5___ pH 10.9 __-2___ c. [5 pts] Suppose this peptide is biologically active only when proline’s peptide bond is a cis-peptide bond. Furthermore, suppose this biologically active peptide is a pharmaceutical target (in other words a money making drug). In addition to standard peptide synthesis, which can be done with a peptide synthesizer, what would be needed in production in order to make the biologically active form? One possible answer: After synthesis, the trans- and cis-form, which exist at a 4:1 ratio of the peptide, would need to be isolated by chromatography. The cis-form would then be ready for use. The trans-form could then be treated with a cis-trans proline isomerase to bring the solution Keq to 4:1. This could be purified to get more of the cis-proline form. This process could be repeated several times. Name ___ANSWERS_____________ Page 7 of 10 3. Protein Heterogeneity [10 pts] List and briefly describe 5 different ways proteins can display heterogeneity. With each of the 5 ways you chose, briefly state how the protein could be purified of the heterogeneity, or describe how each type of heterogeneity could be avoided. Listed below are the 5 most common answers: 1. Polydispersity or oligomerizatoin purify by size exclusion chromatography or solve the problem by adding detergents to coat hydrophobic surface 2. disordered domain reconstruct the expression construct to express individual domains or other reasonable approaches to purify based on a difference 3. Cys oxidation solution, keep in reducing conditions with DTT or beta-mercapto-EtOH 4. ASN/GLN hydrolysis solution: keep at pH 7.0, use immediately and/or store protein at –80 C 5. proteolysis solution: use protease inhibitors during prep or purify by size exclusion chromatography