MHC-I peptide binding activity assessed by exchange after cleavage of peptide covalently linked to β2-microglobulin

A common approach to measuring binding constants involves combining receptor and ligand and measuring the distribution of bound and free states after equilibration. For class I major histocompatibility (MHC-I) proteins, which bind short peptides for presentation to T cells, this approach is precluded by instability of peptide-free protein. Here we develop a method wherein a weakly-binding peptide covalently attached to the N-terminus of the MHC-I β2m subunit is released from the peptide binding site after proteolytic cleavage of the linker. The resultant protein is able to bind added peptide. A direct binding assay and method for estimation of peptide binding constant (K_d) are described, in which fluorescence polarization is used to follow peptide binding. A competition binding assay and method for estimation of inhibitor binding constant (K_i) using the same principle also are also described. The method uses a cubic equation to relate observed binding to probe concentration, probe K_d, inhibitor concentration, and inhibitor K_i under general reaction conditions without assumptions relating to relative binding affinities or concentrations. We also delineate advantages of this approach compared to the Cheng-Prusoff and Munson-Rodbard approaches for estimation of K_i using competition binding data.