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SolutionSet1-3 - Bioc100B Winter 2011 Rubin Problem Set#1...

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Bioc100B Winter 2011 Rubin Problem Set #1 Due Tuesday, January 11 th 4:00 P.M. 1) Size-exclusion chromatography is a technique that can be used to determine approximately the molecular weight of a protein, protein complexes, or simple protein mixtures in solution. You perform size-exclusion chromatography on the same multiple- subunit protein under five different sample conditions and make the following measurements: Sample Solution Conditions Molecular Weight(s) Observed 1 Native buffer (pH 7.4, 150 mM NaCl), 20 ° C 80,000 Da 2 pH 7.4, 1M NaCl, 20 ° C 80,000 Da 3 6M Urea, 20 ° C 50,000 Da, 30,000 Da 4 6M Urea, 10 mM Dithiothreitol, 20 ° C 50,000 Da, 20,000 Da, 10,000 Da 5 Native buffer (pH 7.4, 150 mM NaCl), 65 ° C 50,000 Da, 30,000 Da a) Draw a diagram describing a plausible subunit composition for the protein. Here is one possibility such that the total MW is 80KDa, there are three subunits with the observed MWs, and the interfaces between subunits are consistent with the different experimental results: Note that with the given information, A does not necessarily have to interact directly with both B and C. b) What does comparison between the experiments using Samples #1, #2, and #3 tell you about the interface stabilizing the 50 kDa and 30 kDa subcomplexes? High salt does not disrupt the interface between A and the B-C subcomplex, whereas urea does disrupt this interface. This indicates that the interfaces between A and B-C are hydrophobic in nature and not electrostatic. The fact that B-C remains together as a 30KDa subcomplex even under denaturing conditions (6M Urea) suggests B and C are covalently linked (e.g. through a disulfide bond as suggested by Sample #4, addition of a reducing agent). c) Explain the different thermodynamic mechanisms for subunit dissociation in Samples #3 and #5. Sample 3 contains the denaturant urea. Urea disrupts the hydrophobic effect through its hydrogen bonding with water. The hydrophobic effect lowers the free energy of the native state relative to the unfolded state, so reducing the
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Bioc100B Winter 2011 Rubin hydrophobic effect results in denaturation. In sample 5, the temperature has been raised. High temperature results in denaturation primarily by increasing the weight of the conformational entropy term in the free energy equation (- T
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