title: Determination of receptor-ligand kinetic and equilibrium binding constants using surface plasmon resonance: application to the lck SH2 domain and phosphotyrosyl peptides.
abstract: Experimental and computational methods were developed for surface plasmon resonance (SPR) measurements involving interactions between a solution-binding component and a surface-immobilized ligand. These protocols were used to distinguish differences in affinity between the SH2 domain of lck and phosphotyrosyl peptides. The surface-immobilized ligand was the phosphotyrosyl peptide EPQpYEEIPIA, which contains a consensus sequence (pYEEI) for binding lck SH2. In the kinetic experiment, SPR phenomena were measured during association and dissociation reactions for a series of glutathione-S-transferase (GST)-SH2 concentrations, generating a set of SPR curves. A global computational analysis using an A + B<==>AB model resulted in single set of parameter estimates and statistics. In an abbreviated format, an equilibrium experiment was designed so that equilibrium constants (Keq) could be determined rapidly and accurately. A competitive equilibrium assay was developed for GST-SH2 in which Keq values for a series of phosphotyrosyl peptides (derived from the pYEEI sequence) varied over 3 orders of magnitude. Interestingly, these results highlighted the significance of the +1 glutamate in providing high-affinity binding to the SH2 domain. For most drug discovery programs, these Keq determinations are a sufficient measure of potency for the primary screen, with koff and kon 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.
authors.affiliation: Department of Inflammatory Diseases, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut 06877-0368, USA.
authors.forename: M M
authors.initials: MM
authors.lastname: Morelock
authors.forename: R H
authors.initials: RH
authors.lastname: Ingraham
authors.forename: R
authors.initials: R
authors.lastname: Betageri
authors.forename: S
authors.initials: S
authors.lastname: Jakes
mesh_terms: D000595:Amino Acid Sequence; D001667:Binding, Competitive; D007700:Kinetics; D008024:Ligands; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D019000:Phosphotyrosine; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D013057:Spectrum Analysis / Q000379:methods; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism
chemical_list: D008024:Ligands; D010455:Peptides; D019000:Phosphotyrosine; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)