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 m ajor histocompatibility complex (MHC) class I proteins, but how peptid es bind to class I is not well understood. We used a fluorescence tech nique 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 la beled at their N terminus with dansyl, and binding of dansylated K(d)- restricted peptides to SC-K(d) resulted in significant fluorescence en hancement, which could be inhibited by unmodified K(d)-restricted pept ides. Real-time binding of a dansylated peptide could be followed by m onitoring the fluorescence at 530 nm. The dansylated Plasmodium berghe i circumsporozoite (PbCS) 263-260 peptide bound to ''empty'' SC-K(d) W ith an association rate constant of 1140 M-1s-1, and the subsequent sp ontaneous dissociation of the SC-K(d)-peptide complex was slow. The di ssociation increased dramatically after addition of excess unlabeled P bCS 253-260 peptide, but with a slower association constant for unlabe led 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 pe ptide exchange on K(d) before export to the surface. The apparent acti vation 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-ant ibody interactions.