Determination of Kd in crystal using only crystallographic data look at The First Direct Determination of a Ligand Binding Constant in Protein Crystals Wu SY, Dornan J., Kontopidis G., Taylor P., Walkinshaw M.D. Angew Chem Int Ed Engl, 2001, 40, 582-586.
George ----------------------------------------------------------------------- George Kontopidis Associate Professor of Biochemistry Head of Biochemistry Veterinary School, University of Thessaly Trikalon 224, Karditsa 43100, Greece Tel: +30 24410 66017 Mob: 69 342 643 75 Fax: +30 24410 66041 e-mail: gkontopi...@vet.uth.gr web site: http://www.vet.uth.gr/english/departments_biochemistry.html ----------------------------------------------------------------------- -----Original Message----- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Steven Herron Sent: Monday, June 27, 2011 9:33 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Kd's in Crystals I had success using crystallography to measure the Ca2+ affinity (in the mM range) for a Ca2+ dependent enzyme. See: Characterization and implications of Ca2+ binding to pectate lyase C. Herron SR, Scavetta RD, Garrett M, Legner M, Jurnak F. J Biol Chem. 2003 Apr 4;278(14):12271-7. We measured the occupancy of the Ca2+ ion using three different pH's and 3-4 different Ca2+ concentrations. The presence of the Ca2+ ion altered the conformation of two residues in the binding pocket. In several of the Ca2+ soak experiment the occupancy was between 35% and 70%, where both orientations of the side chains could be modeled separately and their occupancy values refined (see attached picture). We confirmed our crystallographic Kd approach using tryptophan fluorescence. Since it was difficult to measure mM binding affinities using dialysis or titration calorimetry, we turned to crystallography (since we had lots of crystals and beam time). Steve Jacob Keller wrote: >Dear Crystallographers, > >what is the dogma with regard to affinities in crystals? For example, >if I soak three crystals in 1pM, 1nM, and 1uM compound X, and they all >show equivalent density, does that mean that the affinity is really >better than 1pM, or is the crystal of such a high local concentration >(~600mg/mL) that it will be fully occupied at nearly any concentration, >provided external ligand concentration does not change due to binding >in the crystal? I guess there is also the problem that the >crystallization solutions are very non-physiological, but neglecting >that, is there any straightforward way to think of this, or is there a >good reference? > >Jacob Keller > > >
