FIGURE 5.

ISLAD CM and its d-enantiomer interact with the TCR. A, biochemical analysis of peptide interaction with T-cell proteins. Jurkat T-cells were incubated with fluorescently labeled peptides (ISLAD l-CM-Rho, ISLAD d-CM-Rho, ISLAD-Rho, and a TCR d-CP2G-Rho control peptide), cross-linked, and lysed. The T-cell proteins were resolved by SDS-PAGE, and proteins bound to Rho-labeled peptides were detected by the fluorescence of rhodamine (the ladder of protein sizes is indicated in kDa). Subsequently, the gel was transferred to a membrane and subjected to Western blotting for actin. B, Jurkat T-cells were incubated with ISLAD l-CM-Rho, ISLAD d-CM-Rho, and TCR d-CP2G-Rho. The cells were then cross-linked, lysed, and immunoprecipitated with anti-TCRα antibody. Bound proteins were separated by SDS-PAGE and analyzed for the presence of the fluorescently labeled peptides. The results are presented as the mean -fold of fluorescence intensity relative to the control (TCR d-CP2G-Rho) ± S.D. (n = 2). *, p < 0.05. C, fluorescence measurements of peptide-membrane interactions. ISLAD l-CM (□) and ISLAD d-CM (■) were titrated with increasing concentrations of phosphatidylcholine:cholesterol (9:1) LUVs, and changes in the fluorescence anisotropy (arbitrary units (a.u)) of their intrinsic Trp residue were measured. The fitting curve from the nonlinear least-squares model is presented (Equation 1), which indicates the membrane binding affinity constant. D, d-enantiomer CM interaction with the TCRα TMD. FRET experiments used (i) TCR l-CP-NBD and its mutant TCR l-CP2G-NBD as fluorescent donors and (ii) ISLAD l-CM-Rho and ISLAD d-CM-Rho as acceptors. Spectra were obtained with excitation set at 467 nm and an emission scan at 500–600 nm. An NBD-labeled peptide was added to phosphatidylcholine:cholesterol (9:1) LUVs in PBS to a final concentration of 0.1 μm. This was followed by the addition of Rho-labeled peptides in several sequential doses. A nonlinear regression plot of FRET efficiency (arbitrary units) is presented by the relative percentage of emission at 530 nm between the NBD-labeled peptides alone and the peptides at different Rho/NBD-labeled ratios. ▴, ISLAD l-CM-Rho with TCR l-CP-NBD; ■, ISLAD d-CM-Rho with TCR l-CP-NBD; ▵, ISLAD l-CM-Rho with TCR l-CP2G-NBD; □, ISLAD d-CM-Rho with TCR l-CP2G-NBD.