Hi Monica, A different option is they both bind but destabilize your protein (as you suggested). This will likely not help much if you are trying to co-crystallize them. But there are couple of examples out there in the literature that show the contrary, despite destabilizing Tm they got structures with bound ligands - I think it was the Beryllium Clan out in Seattle that had such an example but I might be misremembering this.
There’s a clear dose-dependency in both ligands, so I would give it a shot (assuming you have enough protein & ligands). You could run some ITC or SPR experiments to verify this if you happen to have access to such an instrument. Now if you have an enzymatic assay, that can be tricky if you are looking for inhibition. Yes your ligands may inhibit, great but perhaps your protein is simply destabilized and unfolds etc. and fails to fulfill its normal function - here it would be handy to run some CD spectra to actually se if there is a dose-dependent unfolding of the protein. This will also help you decide how likely it might be to co-crystallize the ligand. Do you have native crystals ? Soak your ligands in and observe if they crack/dissolve. Good luck, Jürgen ...................... Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742<tel:%2B1-410-614-4742> Lab: +1-410-614-4894<tel:%2B1-410-614-4894> Fax: +1-410-955-2926<tel:%2B1-410-955-2926> http://lupo.jhsph.edu On Feb 5, 2015, at 9:33 PM, John Newitt <[email protected]<mailto:[email protected]>> wrote: Hi Monica, Your Tm results don't suggest any binding of your ligand to the protein. Your ligand concentrations are quite high so I am not convinced of even weak binding. The destabilization that you are seeing at the highest concentration may be due to ligand precipitation causing protein denaturation/unfolding. If available, I would search for other ligands for co-crystallization. Alternatively, confirm the ligand binding by some other technique before investing a lot of time and protein in crystallization screening. John Sent from my iPad On Feb 5, 2015, at 8:43 AM, Monica Mittal <[email protected]<mailto:[email protected]>> wrote: Hi all, I am working to crystallize a protein-ligand complex. I did a preliminary melting curve analysis for the protein in the absence and presence of 2 ligands (dissolved in protein buffer). I kept the other controls as buffer an a known standard to confirm instrument performance. All expts done in triplicates. Now the results are like : Tm of protein alone is 56 deg, Tm in the presence of Lig1 conc. of 0.05mM, 0.1mM, 1.0mM and 10.0mM is 56.2, 56.1, 56.0 and 53.5 respectively !! Tm in the presence of Lig 2 conc. of 0.05mM, 0.1mM, 1.0mM, 4.0mM and 10.0mM is 56.2, 56.1, 54.9, 52.3 and 50.6 respectively !! Although the effective delta Tm for both is different at higher concentration, but both are kind of making protein less stable. So i was wondering, will it be difficult to co-crystallize them !! Any suggestions in this regard are highly appreciated !! Thanks Monica
