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Dear Brett, since your deltaH contribution gets weaker, and the deltaS contribution changes from negative to neutral, you could have a case of enthalpy-entropy compensation (see e.g.Dunitz JD (1995) Win some, lose some: enthalpy-entropy compensation in weak intermolecular interactions. *Chem Biol* 2, 709–712). As for the discrepancy between the pull-down and the ITC, remember that a pull-down is not an equilibrium measurement. If you keep washing long enough (hours, days, months), you will eventually lose all binding. The length of time required to wash it off is partly dependent on the kinetics of binding and unbinding. SPR could answer if that's the case. Other things to consider: temperature difference between ITC and pull-down? Cheers Filip Van Petegem -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: [log in to unmask] http://crg.ubc.ca/VanPetegem/ On Tue, Sep 2, 2008 at 1:37 PM, Brett Collins <[log in to unmask]>wrote: > 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 >