12. (20 points) True or False For each statement, indicate whether...

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12. (20 points) True or False For each statement, indicate whether it is True(T) or False (F). If False, make the statement correct. If you find the statement ambiguous, indicate (A) and explain why. a) Proteins with molecular weights smaller than the fractionation range of a gel filtration column will not elute from the column. b) It is not possible to separate two proteins on an ion exchange column if they have the same net charge. c) Two proteins with the same native molecular mass will co-elute from a gel filtration column. d) The two subunits of a protein exhibiting C2 symmetry are mirror images of each other as observed in the intrasubunit isologous contacts. e) A protein with Cs oligomeric symmetry must have five subunits. f) Every residue in a single alpha helix can have different phi and psi angles. g) Doubling the speed (rpm) in a centrifugation experiment will double the rate of sedimentation of a protein. h) All compounds with multiple proton ionizations will have an isoelectric point somewhere between the highest and lowest pKa. i) If a is protein is subjected to mass spectrometry and two peaks are detected, the result indicative of multiple subunits. j) Under standard state conditions, the equilibrium constant for a reaction is 1. k) 2,3-BPG formed induces O2 release from Hemoglobin by binding in a central cationic cavity between the a-subunits of oxyhemoglobin. I) All common amino acids in proteins exist in the S configuration when using the Cahn- Ingold-Prelog or RS system. m) ß-bends are most often found on the surface of proteins and often contain either Gly or Pro residues. n) Just as the a-COOH of one amino acid reacts with the a-NH2 of another residue to form the peptide backbone of a protein, the y-COOH of glutamic acid reacts with the a-NH2 of other residues to make branch points in proteins. o) The structure of collagen is almost purely a-helical with the chains staggered with respect to each other by one full turn. p) Tryptophan and tyrosine account for nearly all of the near UV absorption of proteins and tryptophan absorbs nearly 4 times as much light as tyrosine at 280 nm. q) In an a-helix, the amino acid sidechains (R-groups) point inward where they interact to form a stable tightly packed core. r) The the pl of protein. a protein is calculated as the average of the pKa's of all ionizable groups in s) Protein and folding 4 torsion appears angles. to occur through a random conformational search of the t) Hydropathy on the interior plots are and useful which in predicting which portions of a poly-peptide chain are are on the outside. 13. (5 points) If no precautions are taken, blood that has been stored for some time becomes depleted in 2,3-BPG. What are the possible consequences of using such blood in a transfusion? 14. (10 points) Consider the structure of a water-soluble B-barrel (i.e. not a trans-membrane would and in what distribution (relative to both the B-sheet and the overall etc.) amino acid barrel). residues What general types (eg. hydrophobic, hydrophilic, charged, large, small, of ß- a/ß Justify your answers. barrel you structure? expect to find in this structure? How will your answers change for a water-soluble structure) 15. (10 points) Your company is in the process of developing a biosensor for a commercially important compound, which has been designated HX279-Z. It turns out that a protein, readily available from a variety of sources, is capable of binding this compound. Another group in your company has figured out how to couple that binding to a readily observable spectroscopic signal. Your group is responsible for selecting the best source for the protein for the job. The desired sensitivity is in the 1nM to 100nM range. Your team has assayed the binding of HX279-Z to the protein purified from three different sources - call them A, B and C. The data are presented below. Which is the best choice? Justify your answer. Protein A 1 0.8 0.6 0.4 0.2 2 4 6 8 10 12 [HX279-Z] um Protein B 3.5E+08 3.0E+08 2.5E+08 2.0E+08 1.5E+08 1.0E+08 5.0E+07 0.0E+00 0.2 0.4 0.6 0.8 1 nu-bar Protein C 1 0.8 0.6 0.4 0.2 -10 -9 -8 -7 -6 -5 -4 -3 log( [HX279-Z] in M) 16. (10 points) On one of the early problem sets you were asked to consider the the relationship between the ^Hionization of the buffer (specifically, MES and Tris) and the pH of a solution as a function of temperature. In proteins, the enthalpies of ionization of the R-groups are also important. The table below indicates approximate values for these at 25°C. {For reference, the ^Hionization for MES was about 15 kJ/mole while for Tris it was about 47 kJ/mole.} Group AHionization kJ/mole Asp 3.9 Glu Lys 48 Arg 48 His 28 N-term 40 C-term a) During protein purification, we often work at pH 7 and 4 °C. If we assume that physiological conditions are 37 °C How will the net charge of a typical protein change by lowering the temperature? (You should assume that the pH is controlled by a temperature independent buffer.) b) What can we change to compensate for this difference in charge? c) Briefly consider and discuss whether or not such compensation is necessary? 17 17. following (5 points) reaction: Yeast alcohol dehydrogenase (YADH) is a bisubstrate enzyme that catalyses the NAD+ CH3CH2OH + ethanol CH3COH + NADH H* acetaldehyde This leading enzyme has been shown to obey an ordered sequential Bi-Bi mechanism where NAD* is the product released ). i.e. substrate ( the first substrate bound ) and NADH is the following product ( the last NAD+ EtOH Acetaldehyde NADH If you titrate the enzyme with NAD+ in the absence of ethanol, a plot of fraction YADH with NAD* bound (ie. e vs [NAD*]), allows you to determine the binding constant for NAD* to the enzyme. This experiment works because no reaction occurs without both substrates. If you carried out the reverse experiment (titration of YADH with ethanol in the absence of NAD*), what result or information would you obtain? Why? 18. (10 points) The table below lists the values of kcat and KM for several enzyme substrate pairs. Which of these enzymes, if any, can be considered to have reached "catalytic perfection"? Justify your answer. Enzyme Substrate KM (M) kcat (si) Acetylcholinesterase Acetylcholine 9.5 x 10 5 1.4x104 Carbonic Anhydrase HCO3` 2.6x10² 4.0x105 Catalase H2O2 2.5 x 10-2 1.0x107 Fumarase Malate 2.5 x 10 5 9.0x10² Urease Urea 2.5x10² 1.0x104

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