4. (10 points) You are studying the binding of a large two…
4. (10 points) You are studying the binding of a large two-domain protein to a small molecule inhibitor. Each domain is 60 kDa and they are linked by a 15-amino acid linker. There is a crystal structure of one of domain A showing that it adopts an oblong alpha helical bundle. The second domain (B) does not have a crystal structure, but it is 75% identical to domain A. It has been impossible to obtain a crystal structure of the entire protein with both domains. Finally, there is a controversy of whether the protein is a monomer or dimer in solution. You want to test the hypotheses that: The two domains in the apo (unbound) state are in equilibrium between a V-shaped (closed) and linear (open) conformation. The binding of a small molecule inhibitor causes a large conformational change that favors the closed, V-shape conformation. Although the figure depicts a monomer, it is possible that the apo protein may be in a higher order oligomeric state (e.g., dimer, trimer, etc.). How would you determine the oligomeric state of the protein in the absence and presence of a small molecule ligand? Describe how you would use the scattering techniques you learned about in this class to test ALL THREE parts of your hypothesis. a. (3 points). How would you test if the two domains are in equilibrium between a V-shaped (closed) and linear (open) conformation? b. (3 points). How would you test if the binding of a small molecule inhibitor causes a large conformational change that favors the closed, V-shape conformation? c. (4 points). How would you settle the controversy about the oligomeric state of the protein in the absence and presence of a small molecule ligand?
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