Figure 4.

Peptide binding of DR1 by BIAcore SPR in real time. (a) The binding of DR1_βG86Y to immobilized peptide in real time. Cys-HA_Anchorless peptide was immobilized in a biosensor flow cell and sensograms were obtained by injection of DR1_βG86Y–HA_Y308A complexes (citrate buffer, pH 6.0). The long-term experiments were performed in a BIAcore X instrument (for details, see Materials and Methods). The binding curve is expressed as RU as a function of time. The DR1_βG86Y–HA_Y308A complexes were prepared by preincubation of DR1_βG86Y and HA_Y308A peptide followed by size-exclusion purification. Running buffer was citrate buffer plus 0.01% Tween 20, pH 6.0. One out of two experiments is shown. (b and c) The effects of DM on binding of the wtDR1 (b) and DR1_βG86Y (c) to the peptide surfaces; 2.4 μM wtDR1 or DR1_βG86Y, in complex with HA_Y308A peptide, was isolated by the procedure described in the legend to Fig. 1, and then was incubated at 37°C with or without 1 μM DM for 20 min. Samples (citrate buffer, pH 6.0) were injected over three different peptide surfaces (cys-HA, cys-HA_Anchorless, and the control peptide) of BIAcore 2000 at a flow rate of 4 μl/min for 7 min followed by washout (citrate buffer plus 0.01% Tween 20, pH 6.0) at a flow rate of 5 μl/min. Sensograms of DR1 binding are shown in different colors: in the absence of DM (blue), presence of DM (red), and DM plus soluble peptide for blocking (green). One out of three experiments is shown.