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Dear CCP4 Community, My apologies for the non-crystallography biochemical question but it occurred to me that there are many people on this list who are also very good biochemists. We have just performed an ITC experiment with two proteins and measured a Kd of 150 nM, deltaH of -15 kCal/mol, deltaS of -15 Cal/mol/K and deltaCp of -2000 J/Mol/K. We also measured the binding of a mutant of one of these proteins predicted from crystal structure to inhibit binding of a small fragment of peptide (this is predicted to reduce binding slightly but not to affect total binding as there is still a large interaction interface that is left intact). This mutant has a Kd of 150 nM as well, but deltaH is -10 kCal/mol, delta S is essentially zero, and deltaCp reduces in magnitude to about -1500 J/Mol/K as we would predict from the change of buried surface area. The ITC data looks good and we have repeated the experiments a number of times so they are statistically significant. The experiments were performed within reasonable concentration limits (~10uM protein in the cell so the C-value is about 50-100) Now the puzzle is that the mutant binds less strongly in pulldowns (about 50% reduction after several washes) but we see an almost identical Kd by ITC despite major changes in enthalpy/entropy contributions to binding. The mutant and wildtype appear to have identical fold by CD but of course there may be small differences. Everything makes sense except the lack of Kd change by ITC. Does anyone have any experience of similar results, or more importantly have a possible explanation for them? Any thoughts greatly appreciated. Brett Collins