(a) DR1 molecules complexed to short-lived peptides were allowed to dissociate for various times in the absence or presence DM at pH 5.5 and then pulsed for 1 hour with FITC-HA306–318 at pH 8 (●, + DM; ▴, −DM). Alternatively they were pulsed at pH 5.5 for 1 hour (○) or 2 hours (□). (b) DR1_G86Y-HA_Anchorless (i) or DR1_H81N-HA_306–318 (ii) complexes were allowed to dissociate for various times and pulsed with FITC-HA_306–318 or FITC-HA_Anchorless respectively, for 15 minutes in the absence (○) or presence (▪) of DM. Excess peptide was separated and relative fluorescence from bound FITC-peptide was plotted against the time of dissociation of initial peptide-DR1 complexes. In all cases, the fluorescence intensity at time t₀ was arbitrarily assigned a value of 1.0 and the data at later time points plotted relative to the initial intensity. One of at least two experiments.

(a) DR1 molecules were allowed to bind to FITC-HA_306–318 peptides for 1 hour in the absence (white) or presence of DM in solutions of glycerol (black) or sucrose (grey) of increasing viscosities, made in Citrate Phosphate buffer pH 5.5. *, the “DM effect” (b) The association of FITC-HA_306–318 peptides to DR1 in the absence (○) or presence (▪) of DM was followed over time in i. CP pH 5.5 or viscous solutions of ii. glycerol or iii. sucrose in CP pH 5.5, and fluorescence from bound peptide plotted against time. DR:DM = 5:1 (c, i) DR1 molecules pre-incubated with DM for 5 minutes (black) or 0 minutes (grey) were allowed to bind for 1 hour to FITC-HA_306–318 peptides. The control of 1 hour association without DM (white) is also included. One of at least three experiments. (c, ii) DR1 molecules were either not pre-incubated with DM (white), or were pre-incubated with DM for 5 minutes, followed by removal of DM by immunoprecipitation, IP (black) no removal of DM (grey), and then incubated with fluorescent HA_306–318 for 1 hour. Fluorescence of DR1- HA_306–318 complexes was plotted as histograms. Error bars indicate standard deviations from three experiments.

(a) DM mediated peptide binding is blocked by SEA. 2.4 μM DR1-HA_Y308A complexes were produced, isolated, and incubated with or without 1μM DM or 7μM SEA for 20 minutes, and injected over either HA_306–318 or HA_Anchorless peptide-bound CM5 chip surface in a BIAcore experiment. Samples in Citrate Phosphate (CP) pH 6.0 were injected at 4μL/minute followed by washout (CP pH 6.0 + 0.01% Tween). Raw real-time binding data of DR1 to the peptide chip is shown; in the presence of DM, blue; in the presence of SEA, red; in the presence of DM + SEA, black; in the presence of DM + SEA + EDTA, green. (b) Same as (a), except that SEB or TSST were used instead of SEA. Real-time binding data of DR1 to peptide in the presence of DM, red; SEB + DM, blue; or TSST + DM, black. (c) DM mediated peptide dissociation is blocked by SEA and SEH. 2.4 μM DR1-FITC-HA_Anchorless complexes were allowed to dissociate for 30 minutes in CP pH 5.5, in the presence or absence of 1μM DM, either without SAg (black), or in the presence of 7μM SEA (grey) or 7μM SEH (white). The fluorescence of the complexes is expressed as a fraction of complexes dissociated at 0 minutes, which was assigned an arbitrary value of 1.0. (d) Same as (c), except that the experiment was performed over 20 minutes, and the SAgs used were SEA (grey), SEB (stripes), or a mutant SEA that does not bind β81His (dotted).