REAL-TIME MEASUREMENT OF ANTIGENIC PEPTIDE BINDING TO EMPTY AND PRELOADED SINGLE-CHAIN MAJOR HISTOCOMPATIBILITY COMPLEX CLASS-I MOLECULES

Cytotoxic T lymphocytes (CTL) recognize peptides in association with major histocompatibility complex (MHC) class I proteins, but how peptides bind to class I is not well understood. We used a fluorescence technique to measure antigenic peptide binding to a soluble, single-chain K(d) (SC-K(d)) molecule in which the K(d) heavy chain was connected by a 15-residue link to beta2-microglobulin. Peptides were covalently labeled at their N terminus with dansyl, and binding of dansylated K(d)-restricted peptides to SC-K(d) resulted in significant fluorescence enhancement, which could be inhibited by unmodified K(d)-restricted peptides. Real-time binding of a dansylated peptide could be followed by monitoring the fluorescence at 530 nm. The dansylated Plasmodium berghei circumsporozoite (PbCS) 263-260 peptide bound to ''empty'' SC-K(d) With an association rate constant of 1140 M-1s-1, and the subsequent spontaneous dissociation of the SC-K(d)-peptide complex was slow. The dissociation increased dramatically after addition of excess unlabeled PbCS 253-260 peptide, but with a slower association constant for unlabeled peptide, 77 M-1s-1. Thus, the K(d)-peptide complex on the surface of antigen-presenting cells should be stable, but high concentrations of peptides in the endoplasmic reticulum (ER) lumen would allow for peptide exchange on K(d) before export to the surface. The apparent activation energy for PbCS 253-260 peptide binding to SC-K(d) was 6.78 +/- 0.64 kcal/mole, similar to values previously reported for antigen-antibody interactions.