Reference no: EM132396718

Assignment

1. Imagine that you have just completed the research in papers 5 and 6 (by Zaccardi) and asked to devise the “next experiment”. Generate a new hypothesis and experimental design.  Use a single sheet of white paper and, on the top, clearly write your hypothesis.  Below the hypothesis draw a cartoon/diagram of the experimental design you would use to address your hypothesis.   

What would be the most critical/exciting/innovative/informative experiment you could do? You may choose to follow up on a particular aspect of the paper, recall dangling issues, open questions that require resolution etc.  In your clear diagram, explain what you will do/solve/measure and potential outcomes. The key is to come up with a clear and simple question or a hypothesis and then an experiment that addresses it thoroughly.  Remember – do not try to investigate too much. Pick a question/hypothesis that you can fully answer/test with your design.

2. The discussion for paper 5 states:

“Our proposal is seemingly at odds with the crystal structure of IGP docked into ssIGPS because, in this conformation, the side chains of Glu-51 and Lys-53 are not properly positioned for this type of chemistry.”

Use chimera to generate a figure that shows what this sentence means.  Use arrows and text to annotate you figure to clarify how the figure supports this statement. Use chimera to make the appropriate measurements and display them in your figure.  I would encourage you to make the background white so it is easier to work with your figure.  You can paste it into powerpoint and add annotations there.  It may be helpful to make multiple frames/views, in other words, to rotate the structure so that y you can show what you want to show.  The pdb code for the structure you need can be found in papers 4 and 5.
 
3a. Take a look at paper 5, figure 4B.  This is the revised mechanism for SsIGPS catalyzed reaction. If you had to pick one thing about this mechanism that you do not like/that you think might be wrong, what would it be?  Draw your updated mechanism and circle the place where you modified the mechanism.  Finally, in one or two concise sentence/s, explain why you modified the mechanism.  If you think the mechanism is the best possible mechanism, what aspect of it is not fully proved?  What would be an alternative possibility for that aspect?

3b. Look at paper 5, figure 4B again.  If we assume this is the correct mechanism, draw a substrate/product analog (something that has a structure with some similarities to the substrate or product) that you think would be a good candidate for inhibitor for this enzyme.  In other words, look at the mechanism and interactions and think about what you could change to make the substrate or product such that it would still bind well but not react. Draw the structure of your proposed inhibitor.

4. Look at Paper 6 (Zaccardi 2014) Figure 2.  Based on the paper and figure, draw the Lys53 side chain in its predominant ionization state at pH values 3.0 and 9.0 for both the wildtype enzyme and the N90A enzymes.

5. Let’s say that you had to reconsider the mechanism shown in Paper 5, figure 4B and you had to come up with an alternative hypothesis for a residue that would act as the catalytic base in the dehydration step.  Use what you know about IGPS and chimera to propose another possible amino acid (other than Glu51) to play this role.  A few hints: the residue needs to be able to serve as a base, be  conserved,  and be located sufficiently close to the site of deprotonation in this step. As we know, there is movement during the catalytic cycle, so even if the structure shows the residue further that what seems realistic, it may move in during the critical moment.  State the residue you propose to serve this role instead of Glu51 and provide a chimera figure to support your idea.

6. Draw a detailed and labeled diagram to show how the authors determined whether there was a viscosity effect for a particular mutant IGPS E210Q.  Be sure to draw a diagram/cartoon rather than write text.  Only a diagram will receive points. Assume that you have already all the mutant and the substrate available.  You do not need to show how they were produced.  Please, show the steps of the experiment, what the raw data looked like and how it was analyzed to determine the answer shown in table 1 in paper 5. Your figure can take up 1 page.

7.  Use chimera to illustrate the interaction described in paper 6: “we examined the role of the invariant Phe89 residue located on the beta2alpha2 loop. This residue makes a π– π interaction with the anthranilate moiety of CdRP.  Please, show whether this interaction is equally present in the CdRP, rCdRP and IGP structures.

8.  Paper 6 indicates that the rate determining step for the wildtype enzyme is different at 37 degrees C and at 75 degrees C.  What is the rate determining step at each temperature?  Why do you think the rate limiting step is changing, what could explain this?

9. Given the data in Table 1 of paper 5, show how the authors calculated the double mutant cycle to make the conclusion that coupling was present.  You don’t need to show how to calculate ΔΔG here because that is already shown in the table.  Just show how they analyzed the values to identify coupling.

Reading:

The Catalytic Mechanism of Indole-3-glycerol Phosphate Synthase: Crystal Structures of Complexes of the Enzyme from Sulfolobus solfataricus with Substrate Analogue, Substrate, and Product By M.Hennig, B.D.Darimont, J.N. Jansonius and K. Kirschner.

Functional Identification of the General Acid and Base in the Dehydration Step of Indole-3-glycerol Phosphate Synthase Catalysis By Margot J. Zaccardi and Eric M. Yezdimer.

Loop-loop interactions govern multiple steps in indole-3-glycerol phosphate synthase catalysis By Margot J. Zaccardi and Kathleen F. O’Rourke.