SolutionSet1-2

SolutionSet1-2 - Bioc100B Winter 2010 Rubin Solution Set #1...

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Bioc100B Winter 2010 Rubin Solution Set #1 1) Size-exclusion chromatography is a technique that can be used to determine approximately the molecular weight of a protein, protein complex, or simple protein mixture in solution. You perform size-exclusion chromatography on the same 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 100,000 Da 2 pH 7.4, 1M NaCl, 20 ° C 60,000 Da, 40,000 Da 3 6M Urea, 20 ° C 60,000 Da, 20,000 Da 4 6M Urea, 10 mM 2-mercaptoethanol, 20 ° C 50,000 Da, 20,000 Da, 10,000 Da 5 10 mM 2-mercaptoethanol, 65 ° C 50,000 Da, 20,000 Da, 10,000 Da a) Draw a diagram describing a plausible subunit composition for the protein. Here is one possibility such that the total MW is 100KDa, there are four subunits with the observed MWs, and the interfaces between subunits are consistent with the different experimental results: b) What does the experiment tells you about the forces that stabilize the different interfaces between the subunits within the quaternary structure? 1M NaCl dissociates the complex into a 60kDa fragment (A-C) and a 40kDa fragment (B-B’). This result suggests that the interface between these two subcomplexes is stabilized by electrostatic interactions that are weakened in the presence of high ion concentrations. The interface between B and B’ is destabilized by urea or high temperature suggesting it is a hydrophobic interface. A and C remain in complex even under denaturing conditions, but are dissociated in the presence of a reducing agent. This observation indicates that A and C are covalently linked by a disulfide bond.
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Bioc100B Winter 2010 Rubin c) Explain the different thermodynamic mechanisms for subunit dissociation in samples 4 and 5. Sample 4 contains the denaturant urea.
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This note was uploaded on 03/16/2010 for the course BIOC 100B taught by Professor Sethrubin during the Winter '10 term at UCSC.

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SolutionSet1-2 - Bioc100B Winter 2010 Rubin Solution Set #1...

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