Figure 4.

Native gp100_209–217–MHC-I complex is metastable. (A and B) Determination of peptide dissociation rates from target cell surface. Calculated amounts of gp100_209–217 (filled squares) and gp100_{209–217(2M)} (open squares) peptide bound to HLA-A2 molecules were plotted over time. Apparent k_off values were determined by fitting to a single exponential decay of all points above an undetectable concentration of peptide (1 × 10^–10 M using human T cell clone for A and 1 × 10^–9 M using JR209-Tg T cells for B). t_1/2 was determined from the relationship t_1/2 = 0.693/k_off. (C) Direct assay of peptide dissociation from purified peptide-MHC complexes using fluorescence anisotropy. Dissociation rates and t_1/2of gp100_209–217and gp100_{209–217(2M)} peptides are indicated. Experiments were repeated in triplicate; experimental errors for the reported parameters were 3% for gp100_209–217 and 7% for gp100_{209–217(2M)}. mA, millianisotropy.