Photocleavable peptides with reduced interaction with DR1 P1 pocket generate DR1_a independently of UV exposure.

<p>(A) The ability of L1MxN and V1MxN peptides to bind DR1 was measured in a competition binding assay using labeled HA. Different concentrations of photocleavable peptides or unlabeled HA were incubated for 3 days with a 150 nM DR1 and 25 nM labeled HA. Binding of the labeled peptide to DR1 was quantified by fluorescence polarization and the fraction of binding that was inhibited by the unlabeled peptide was calculated and plotted against unlabeled peptide concentration. Squares, circles and triangles show the mean and the error bars represent the standard deviation of two independent experiments done with triplicate samples. (B) DR1-L1MxN (left panel) and DR1-V1MxN (right panel) were not exposed (grey trace) or exposed 5 (light blue) and 20 minutes (blue trace) to UV light on ice. Then, a 150 nM of each sample was mixed with 25 nM labeled HA and binding of the labeled HA to DR1 was quantified by fluorescence polarization. Concentration of the labeled peptide bound to DR1 is plotted against time. Each data point shown in the plot is the mean of duplicate samples from one of two independent experiments. (C) Initial peptide binding rates were calculated as the slope of a linear fit to the initial time points of the peptide binding data shown in panel B and plotted as a bar graph. The bars represent the mean and the error bars the standard deviation of the initial rates calculated from two independent experiments done with duplicate samples.