Protein structure and ligand binding

Question

frostbite · Answer

Not long ago, my lab group tried to resolve the structure of human glutaredoxin (a redox enzyme), using NMR spectroscopy and the structure is found in attachment 1. The side chains have been shown for Cys23, Tyr25 and Tyr60 and we found that Cys23 is in the active site.

a) Draw what the Ramachandran plot would be expected to look like based on the structure solved from NMR spectroscopy.

b) Describe the stabilizing interactions in $\beta$-sheets, followed by what amino acid residues we expect in the $\beta$-sheets with explanation for the residue choice.

While doing our characterization, we found that reduced glutaredoxin is been competitively inhibited by high concentrations of glutathione (a tripeptide with the sequence $\gamma$Glu-Cys-Gly). The binding of glutathione to glutaredoxin has been measured using fluorescence spectroscopy and the results are shown in attachment 2. The protein concentration used in the experiment was 50 $\mu$M

c) Which of the two tyrosine residues, would we expect to give the highest change in fluorescence when glutathione binds?

d) Calculate the dissociation constant ($K_{d}$) and binding energy ($\Delta G^{\Theta}_{bind}$) and the stoichiometry in the bond.

We choose to determine the stability of reduced glutaredoxin in the presence and absence of glutathione by doing a titration with urea. The results are shown in attachment 3. When we did this experiment, one in the lab group forgot to label the sample so we got an extra question.

e) Which of the two stability curves is from glutaredoxin in presence of glutathione and why?

frostbite · Answer

The data in attachment two has been modified a little to help the one solving the questions.

anonymous · Answer

Nice problem! Thanks for making all that stuff up, I can feel how much fun you had =)

a) I can only guess ramachandran plots, but I have "drawn" one in my attachments. The Protein consists of helical structures that flank a parallel and an antiparallel beta-sheet.

b) Since the helical structures block water from reaching the beta sheets, I would expect hydrophobic interactions to be the main driver of folding. You always get hydrogen bonds in the peptide backbone that contributes to interaction, but I guess that a fully folded protein almost always has some kind of hydrophobic core. 

c) I expect Tyr25 to get quenched, possibly by interaction with the tripeptide's glutamate. I expect the cysteine to form a disulfide bond with the active center, and spacial proximity does the rest. I would not count that under "competitive inhibition", though...it just binds to the protein and inhibits it reversibly. Wouldn't competitive inhibition be characterized by glutathione binding to the target of glutaredoxine?

d) I am no expert in Kd calculation. Assuming that you provided all cysteines and methionines that are present in the glutaredoxine, the stochiometry is 1, making it a simple reaction of A + B <=> AB. In this case, Kd should value around 15 mM - this is only an estimate, though, since my excel skills are done for today, I couldn't simulate the reaction properly =) Binding energy remains to be calculated! Something with 1/Kd * e ^ (RT) or something? ^^

e) From my previous thoughts about cystine formation and Tyr25 quenching, I expect a more rigid structure of the protein in presence of glutathione. Urea interacts with the amide backbone when unfolding a protein, so my estimates are possibly wrong. I still think that the triangles are glutaredoxine without glutathione, while the balls are glutaredoxine in the presence of glutathione.

frostbite · Answer

All of it is correct. The $\Delta G^{\Theta}_{bind}$ I believe you meant the relation:
$$\Large K_{d}=\exp \left( \frac{ \Delta G^\Theta_{bind}}{ RT} \right)$$
Remember that $K_d$ is the dissociation constant, so if we use that constant it must follow the dissociation energy is the negative of the bind energy, which is the energy we want. So we get the relation
$$\Large \Delta G^\Theta_{bind}=RT \ln(K_d)$$

frostbite · Answer

The other info in d) can be extracted from a Scatchard analysis.
But well done!

anonymous · Answer

Thanks! \o/

freethinker · Answer

why are you using Gibbs-Free for binding energy?

frostbite · Answer

It is because we can relate the dissociation constant to the free energy during equilibrium and tells us about the affinity for the substrate.