BMB170c_2011_PS1 - BMB/Bi/Ch 170c 2010 Problem Set 1:...

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Unformatted text preview: BMB/Bi/Ch 170c 2010 Problem Set 1: Regulated Proteolysis Due: April 12, 2011 1. In 2000, Thrower et. al published their findings regarding substrate recognition by the 26S proteosome. Read the paper carefully and answer the following questions (25 points total). a. What are the major conclusions of this paper? (9 points) b. Table I shows that Ub4 is the minimum signal recognized by the proteosome. However, it is apparent that Ubn chains with n>4 bind more tightly to the proteosome. The authors provided two models that can explain this observation. What are these two models? Describe the experiment(s) that they performed to resolve which model is correct. State the results (10 points). c. The authors conclude that protein degradation is rate ­limited by the unfolding of substrate. Describe the experimental results that led to this conclusion. Refer to figures in the paper (6 points). 2. Kenniston, et. al (2003) studied the mechanism by which ClpXP degrades its protein substrates (25 points total). a. The authors chose Titin I27 as their degradation substrate to test their hypotheses. What are the properties of Titin I27 that made it a good candidate to study in relation to their hypotheses? (7 points) b. Mutations introduced to Titin I27 altered the amount of ATP consumed per molecule of substrate. However, the binding affinities of these ssrA ­tagged mutants remain the same. Which step(s) of the protein degradation pathway account(s) for the difference in ATP consumption? Why are the Km values unaffected when substrate stability is increased/decreased? (8 points) c. A hypothetical protein composed of 120 amino acids (comparable length to Titin I27) has ∆Gu of 7 kcal/mole. When an ssrA tag is attached to the C ­terminal end of this protein, ClpXP degrades it at an overall rate of kdeg = 0.7 min ­1. Based on the results in this paper, estimate, for this hypothetical substrate, the binding affinity to ClpXP, the rate of translocation ktrans, the rate of denaturation kden, the number of ATP hydrolyzed per molecule degraded, and the ATPase rate. Show your work/reasoning. (10 points). 3. Kisselev, et. al (2006) performed experiments that tested the effect of inhibitor(s) on the peptidase activities of the 26S proteosome (25 points total). a. Briefly summarize the major conclusions of this paper (7 points) b. The authors used a variety of inhibitors that targeted different active sites in the proteosomal core. Table 3 shows that the %inhibition of degradation by these inhibitors significantly varies with different protein substrates. Provide an explanation that accounts for this observation (8 points). c. For your rotation project, you were asked to design an inhibitor of the proteosome. How will you approach this task? Which site(s) on the proteosome will you target and why? Why is your inhibitor better than the ones used in this paper? (10 points) 4. In 2007 and 2008, the Goldberg and Cheng labs published their studies regarding the structural basis and mechanism of gate opening in the archaeal 20S proteosome triggered by the regulatory PAN ATPase complex. (Smith, et. al; Rabl et. al. (25 points total). a. The proteasome regulators PA26 and PA28 were shown to use their ‘activation domains’ that work in concert with their C ­termini to facilitate gate opening in the 20S. Do the PAN and 19S regulatory particles employ the same mechanism? What is the evidence? (10 points). b. The Cryo ­EM structures of the open ­ and the closed ­gate forms of the archaeal 20S was solved by Rabl et. al. How were the experimenters able to trap the open ­ gate forms? What are the key residues in the 20S that interact with the gate ­opening peptides? (8 points). c. Describe the structural basis for gate ­opening by PAN that was proposed by the authors based on the structures that they solved. Was the resolution of the structure sufficient to make this conclusion? (7 points). ...
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