DETERMINATION OF RECEPTOR-LIGAND KINETIC AND EQUILIBRIUM BINDING CONSTANTS USING SURFACE-PLASMON RESONANCE - APPLICATION TO THE LCK SH2 DOMAIN AND PHOSPHOTYROSYL PEPTIDES

Experimental and computational methods were developed for surface plas mon resonance (SPR) measurements involving interactions between a solu tion-binding component and a surface-immobilized ligand. These protoco ls were used to distinguish differences in affinity between the SH2 do main of lck and phosphotyrosyl peptides. The surface-immobilized ligan d was the phosphotyrosyl peptide EPQpYEEIPIA, which contains a consens us sequence (pYEEI) for binding lck SH2. In the kinetic experiment, SP R phenomena were measured during association and dissociation reaction s for a series of glutathione-S-transferase (GST)-SH2 concentrations, generating a set of SPR curves. A global computational analysis using an A + B double left right arrow AB model resulted in single set of pa rameter estimates and statistics. In an abbreviated format, an equilib rium experiment was designed so that equilibrium constants (K-eq) coul d be determined rapidly and accurately. A competitive equilibrium assa y was developed for GST-SH2 in which K-eq values for a series of phosp hotyrosyl peptides (derived from the pYEEI sequence) varied over 3 ord ers of magnitude. Interestingly, these results highlighted the signifi cance of the +1 glutamate in providing high-affinity binding to the SH 2 domain. For most drug discovery programs, these K-eq determinations are a sufficient measure of potency for the primary screen, with k(off ) and k(on) determined in a secondary assay. Thus, the application of these techniques to SPR binding phenomena should prove valuable in the discovery and design of receptor-ligand antagonists.