2007/9/11 4:53:00 PM The Second Qualification Exam (2006) for NTHU PART 1 1. The structural motifs formed by the association of two or more helices are categorized as supersecondary structures. Draw the following supersecondary structures in proteins. (1). -, (2). --, (3) hairpin, and (4) Greek key. (8%) 2. Name four of the bonds between two atoms from the strongest to the weakest. (4%) 3. Define the following terms (1) protein domain, (2) protein fold, and (3) protein quaternary structure. (3%) PART 2 1. If you intend to express a foreign protein for the assay of its function, what is your concern? For example, what are the expression systems that will be used and what is your reasoning; do you have to consider posttranslational modifications? If so, what should you do, and so on so forth? PART 3 1. Use proton as an example, please describe the occurrence of NMR briefly. (6%) 2. Please define briefly the following terms. (a) Spin echo (b) Spin-lattice relaxation time (c) Nuclear Overhauser enhancement (d) Free induction decay (e) Pulse sequence (f) Solvent suppression (g) Magic angle spinning 1 (2% each) 2007/9/11 4:53:00 PM PART 4 1. A constructive interference (reflection) is seen only when Bragg’s equation (or Bragg’s law) is satisfied for a particular set of Bragg planes. Please use figure and equation to explain the Bragg’s law. (5 point) 2. For Fourier synthesis to work, we need to know the phases for all reflections. For a long time, obtaining phases was one of the rate limiting steps in crystal structure determination. To date, there are several methods to solve the phase problem. Please list three major methods in the phase determination of the protein crystallography and explain the principle, requirement and the limitation in the each method. (10 pts) .3. (a) What is anomalous scattering? (b) Please use a diagram and equation to explain why anomalous scattering can breakdown the Fridel’s law. (5 point) PART 5 1. 2D gel electrophoresis (2-DE) is the most commonly used separation technique today, (1) please describe the separation principle (2) Can you use the following example to draw a representative 2DE map for comparative experiment. Example, you want to look for proteins that are differentially expressed in Hela cell after stimulation of growth factor (10 points) 2. For identification of protein, mass spectrometry has become one of the core technologies for unknown protein identification. Please describe one of the following principles: (a) Peptide Mass Fingerprinting or (b) Tandem mass spectrometry. (5 points) 2 2007/9/11 4:53:00 PM PART 6 1. As we know that the native tertiary structure of proteins/enzymes is stabilized by number factors. Please (a) provide the names and chemical structures of the four amino acid side-chains involved in “salt bridges”. (4 points) (b) elucidate the identity of the singular covalent “cross link” that is occasionally found. Why do you think Mother Nature had to resort to the use of such linkages? (2 points) 2. Match each statement below with the most relevant concept by placing the appropriate letter in the boxes. (6 points) A Induced-fit theory B kcat C Zipper principle (a) Represents the maximum number of substrate molecules converted to product molecules per active site per unit of time, that is, the number of times the enzyme “turns over” the substrate to the product per unit of time. (b) Binding of substrate triggers a change in enzyme conformation from a catalytically inactive to a catalytically active one. (c) Stepwise binding of flexible, high molecular weight ligands to their receptors. 3. Consider a particular zinc carboxydipeptidase (cleaves dipeptides from the C-terminus of proteins), which specifically recognizes a tyrosine residue at the penultimate position. Some measured kinetics parameters for the “specific substrate” N-(acetyl)glycyl-L-tyrosyl-L-valine (1) and some relatively poor substrate analogues, as well as for some inhibitors are listed below. Km (mM) 3 Vmax (sec-1) 2007/9/11 4:53:00 PM Ac-Gly-L-Tyr-L-Val-OH (1) 0.70 Ac-Gly-L-Ala-L-Val-OH (2) Ac-Gly-L-Tyr-L-Val-NH2 (3) 0.40 5.0 3500 120 2100 Ki (mM) Ac-NHCH2SO2-L-Tyr-L-Val-OH (4) Ac-NHCH2SO2-L-Tyr-L-Val-NH2 (5) 0.03 0.41 no turnover no turnover (a) What factors do you think rationalize why 2 and 3 are poor substrates relative to 1? (Recall that “goodness” of a substrate is represented by V max/Km and other specific interactions as well as IBE) (3 points) (b) Why are 4 and 5 good inhibitors? (2 points) (c) Based on the molecules shown above, you might go about raising monoclonal antibodies which catalyze the same reaction as above with the exception that dipeptides are cleaved specifically when lysine rather than tyrosine is present at the penultimate position. Please provide the appropriate structure of the ideal competitive inhibitor of your antibody catalyst. (2 points) (d) In the Michaelis-Menton plot below, the uninhibited reaction is described by curve 1. The effect of a competitive inhibitor is represented by curve: (1 points) 4