Hello John, No, they're not. Crystals were obtained at pH8.0, 200mM NaCl; 10% PEG4000. Calorimetric experiments were done at pH7.4, 150mM KCl. We found the interaction to be driven mainly by hydrophobic contacts (mutants of polar/charged residues have no significant effect on the affinity). I'd only expect a minor effect of pH in this case, but this would have to be tested. So yes, as I've mentioned before, both crystal contacts and crystallization conditions could together reduce the affinity and break the 100nM Kd interaction.
The effect of pH on the affinity could be tested directly in an ITC experiment - the effect of 10% PEG4000 would be harder to assess due to insolubility (crystallizability) of the protein... Cheers Filip Van Petegem On Mon, Jun 30, 2008 at 4:47 PM, John A. Newitt <[EMAIL PROTECTED]> wrote: > At 3:28 PM -0700 6/30/08, Filip Van Petegem wrote: > > The crystal artefact is that we don't observe any binding in the crystal >> structures of a set of mutants (neither to the native site, nor to any >> other), whereas both calorimetric and electrophysiological data suggest >> there should be binding in the 100-200nM range. The binding is abolished >> because of crystal contacts (+ crystallization conditions) for 100nM and >> weaker binders, but not for 10nM and stronger binders. >> > > Filip: > > Are you calorimetric binding measurements performed under similar > conditions (especially pH) as your crystallization condition for the mutant > proteins? We have determined in some cases that apo crystals are due to the > fact that a ligand had reduced affinity at the non-neutral pH of > crystallization, whereas initial positive binding studies were performed at > pH ~7. > > - John > -- > <http://xri.net/=john.newitt> > -- 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: [EMAIL PROTECTED] http://crg.ubc.ca/VanPetegem/
