A, peptides based on the MBP_85–99 sequence were synthesized to contain a nonproteinogenic amino acid (Anp, blue circles) that undergoes photocleavage when exposed to 365-nm UV light. MBP-488 is the reporter peptide used throughout, and it carries an Alexa-488 at the P5 Cys residue. MBP-C647 and MBP-N647 are peptides onto which Alexa-647 is appended C- or N-terminally to photocleavable MBP-P4* and were used to monitor the departure of cleavage fragments from DR2. B, model of MBP-P4* peptide and major cleavage products formed upon exposure to UV light. Cleavage occurs between the P3 and P4 positions, thus separating the major DR2 anchor residues (P1 Val and P4 Phe) (30).

A, photocleavable peptides MBP-P1* to MBP-P5* were examined for their ability to compete with MBP-488 (10 nm) for DR2 binding (100 nm). MBP-P4* bound most tightly. B and C, photocleavage of MBP-P4* was rapid both in solution or bound to DR2. D, mass spectrometry of intact peptide (top panel, 2+ and 3+ ions) and photocleaved peptide (bottom panel, 1+ ions) showing major cleavage products of molecular weights 1002.6 (C-terminal fragment) and 1091.6 (N-terminal fragment).

Different DR2-peptide complexes (150 nm) were left unilluminated (−UV) or exposed to UV light (+UV) in the presence of MBP-488 (30 nm) for 2 min. Association of MBP-488 was followed by FP after photocleavage. A, rapid association of MBP-488 occurs upon UV irradiation of DR2-MBP-P4*, but not in control reactions. B and C, high affinity MBP_85–99 peptide could not be displaced (B), and slow binding to DR2 molecules with the lower affinity CLIP peptide (C) was observed. D, the rate of MBP-488 association was compared for reactions with and without DM. DM was present during photocleavage at an equimolar concentration to DR2, and MBP-488 was added after photocleavage.

A, DR2-MBP-P4* (160 nm) was photocleaved for 10 min on ice in the presence (red line) or absence (blue line) of MBP-488 (220 nm). When peptide was present during photocleavage, the reaction proceeded rapidly (10 min on ice) to ~100 mP, whereas peptide association was slower when the peptide was added following photocleavage (<100 FP after 50 min). Time 0 is defined by the addition of MBP-488 following photocleavage (blue line). B, dose-dependent acceleration of peptide association to empty DR2. DM was present at different concentrations (150 nm to 5 µm) during photocleavage (10 min, 160 nm DR2-MBP-P4*), and MBP-488 was added (330 nm) after photocleavage. Saturation was not observed because of the lower affinity of soluble DM for soluble DR compared with full-length molecules (42).

A, photocleaved DR2-MBP-P4* (160 nm) ± DM (160 nm) was injected into a cuvette containing 220 nm MBP-488 to monitor the early stages of the peptide binding reaction by FP, immediately upon the addition of DR2. When DM was present during photocleavage, a rapid burst of binding was observed. The rate of MBP-488 association was intermediate when DM was added following photocleavage. Association in the absence of DM was slower. B, incubation of DM with empty DR2 restores and maintains the highly active DR2 conformer. DR2-MBP-P4* was photocleaved for 10 min in the absence of DM. Association of MBP-488 was then compared between reactions with or without DM (1 µm). After different preincubation times (0–2 h), the DR/DM mix or DR alone was injected into a cuvette containing 50 nm MBP-488, and the FP values were monitored continuously.

DR2-MBP-P4* was photocleaved for 5 min, and peptide fragments were removed from DR2 on a HPLC gel filtration column. A and B, the contribution of DM (1 µm) to the kinetics of MBP-488 (1 µm) association was compared between reactions with photocleaved DR2-MBP-P4* (1 µm) from which cleavage fragments had been removed (B) or not removed (A) by Superose 6 size exclusion chromatography. C and D, cleavage fragments added back at different concentrations (D) were without effect on reactions with DR2 molecules not subjected to gel filtration (C) at concentrations of 150 nm DR2-MBP-P4*, 150 nm MBP-488, and 1 µm DM. D, photocleaved peptide fragments were added back at concentrations ranging from 15 nm to 150 nm (DR2 at 150 nm) following purification of DR2 by size exclusion chromatography. The cleavage fragments also did not affect association of MBP-488 in the absence of DM (−DM).

A, peptide-loaded MHC (pMHC), empty MHC (eMHC), and fluorescent peptide-loaded MHC (fpMHC) are shown in the absence of DM (top row) and interacting with DM (bottom row). Peptide photocleavage creates a short-lived MHC molecule with bound cleavage fragments (p^*MHC), whereas incubation in the absence of peptide creates an inactive form (iMHC). eMHC remains in a peptide-receptive form only for short periods of time, but eMHC bound in the DM-eMHC complex (boxed) remains active for extended periods of time. B, the hypothesis represented by Equation 2 fits the experimental data without DM very well. The initial concentration of pMHC was 150 nm, and the amount of MBP-488 added after 10 min of UV was 200 nm. C, the model represented by Equation 3 fits the experimental data well in the presence of variable amounts of DM. The initial concentration of pMHC was 150 nm, and the amount of MBP-488 added after 10 min of UV was 200 nm. DM addition occurred at the same time as MBP-488 addition. A summary of the concordance between experimental data and the kinetic model for conditions not shown here is provided in the supplemental information. Because of uncertainties associated with the length of time required to add MBP-488 and to take the first reading, it was assumed that 30 s elapsed during this process for all DM experiments. Because the no DM data were read last, it was assumed that 2 min elapsed before the first reading. The values of the delay times were chosen to improve the quality of the fit. We also carried out the analyses assuming that the elapsed times were negligible. None of the qualitative conclusions changed upon making this assumption; only the quantitative comparisons with experimental data were less precise at short times.