CALORIMETRIC INVESTIGATION OF PROTON LINKAGE BY MONITORING BOTH THE ENTHALPY AND ASSOCIATION CONSTANT OF BINDING - APPLICATION TO THE INTERACTION OF THE SRC SH2 DOMAIN WITH A HIGH-AFFINITY TYROSYL PHOSPHOPEPTIDE

The binding of Src homology 2 (SH2) domains to tyrosyl phosphopeptides depends on electrostatic interactions between the phosphotyrosine and its binding site. To probe the role of these interactions, we have us ed isothermal titration calorimetry to study the pH dependence of the binding of the SH2 domain of the Src kinase to a high-affinity tyrosyl phosphopeptide. Two independent approaches were employed. In a first series of experiments that focused on determining the peptide's associ ation constant between pH 5.0 and 9.0, two ionizable groups were chara cterized. One group, with free and bound pK(a)s of 6.2 and 4,4, respec tively, could be identified as the phosphate in the phosphotyrosine wh ile the ether group, with free and bound pK(a)s of 8.2 and 8.5, respec tively, could be only tentatively assigned to a cysteine in the phosph otyrosine binding pocket. Further information on the linkage between p eptide binding and protonation of the phosphotyrosine was obtained fro m a second series of experiments, which focused on determining the pep tide binding enthalpy at low values of pH in several buffers with diff erent ionization enthalpies. These data provided free and bound pK(a) values for the phosphotyrosine identical to those derived from the fir st series of experiments, and hence demonstrated for the first time th at the two approaches provide identical information regarding proton l inkage. In addition, the second series of experiments also determined the intrinsic enthalpy of binding of both the protonated and deprotona ted phosphate forms of the peptide. These two sets of experiments prov ided a complete energetic profile of the linkage between phosphate ion ization and peptide binding. From this profile, it was determined that the PO32- form of the peptide binds 2.3 kcal mol(-1) more favorably t han the PO3H1-form due entirely to a more favorable entropy of binding .