We are working on a protein of hyperthermophilic origin that remains in dimeric state at native pH ( pH 8.0). It dissociates into folded monomers at pH 2.0.
The protein is kinetically stable in dimeric state (at pH 8.0) as it exhibits hysteresis in equilibrium unfolding experiment, i.e. normalized unfolding and refolding curves were non overlapping after overnight incubation. Only after incubation at 60 Deg C for 5 days, did refolding curve shift towards lower denaturant concentration and eventually overlapped with the refolding trace. Therefore, the dimer exhibits kinetic stability towards unfolding.
Limited proteolysis is an another measure of kinetic stability of a protein, therefore we attempted limited proteolysis of dimeric (pH 8.0) and monomeric version (pH 2.0) with trypsin and pepsin, respectively. The dimeric protein was resistant to limited proteolysis whereas monomeric version was susceptible to it. The problem is however, was the different types of proteases used at pH 8.0 and pH 2.0. Even if any protease is active at both the pHs, its activity will vary at such a wide pH range which will again raise the question about the comparison. How can we compare the protease sensitivity in this case?
This is really neat - did you run a gel on your partial digests - should produce a peptide map that that provides lots of information concerning protease sensitive sites. shifting time, pH and Protease's should elucidate even more structural information because the map will change. All you need is a set of conditions that gives you reproducible results from which you can proceed. Knowing a little more about protein sequence and some specific folding would give you addition hints as to where sensitive/exposed sites are located. Knowing the protease specificity and how they match with those exposed sites gives you a shot at predicting fragment lengths.
Love to see your data as you progress -
Best Regards
Dan
Dear Dushyant,
I have a few questions before I attempt an answer:
1) When you do equilibrium unfolding with a chaotrope, do you see two trasitions corresponding to dimer-monomer transition and monomer unfolding transition? If no, what is the composite Cm for Urea and GndHCl? Did you attempt temperature unfolding and refolding with, say, circular dichroism and do you still see hysteretic behavior? What is the Tm of the unfolding? The reason I ask this question is that I am confused that a stable dimer unfolds at lower chaotrope while the monomer refolds at higher chaotrope concentration. An idea about the magnitude of concentration difference of the chaotrope for the unfolding and refolding transitions would be helpful in arriving at some insight.
2) Are you doing gel filtration to assess the oligomeric status of your protein across the two pH values? If you have a structure, what kind of residues populate the interface and do you see some residue/s that might be critical for the dimer formation as they get deprotonated at higher pH. If you indeed have such a residue/s, rather than adopting an approach which might require you to resort to gel-based separation and subsequent MS with its attendant problems of interpretational mosaicity, you can simply mutate the residue/s to get a constitutive monomer that will make your analysis more straightforward.
Best
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