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 β₂‐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.