( C) Average dissociation rate constants of uncatalyzed and catalyzed peptide dissociation from H2-D b, using the same conditions as in ( B). The arrow indicates the addition of a 1000-fold molar excess of unlabeled high-affinity competitor peptide (ASNENMETM) without TAPBPR (black trace) or in combination with 1 µM TAPBPR (red, blue, and yellow traces). ( B) Peptide dissociation kinetics from H2-D b (300 nM) loaded with fluorescently-labeled peptide (TQSC*NTQSI) was monitored in real time by fluorescence polarization. ( A) Schematic of peptide displacement assay. ( C) Stability of complexes formed by TAPBPR wt, TAPBPR Tsn-SL, and TAPBPR ΔSL, respectively, as judged by the area of the complex peak obtained by deconvolution. The residual plot depicted beneath the main panel shows the difference between the experimental data and the sum. The sum of the three Gaussians is shown as dotted curve. The experimental chromatogram (red) was deconvoluted using three Gaussian functions (gray) that can be ascribed to the TAPBPR-H2-D b complex (1.06 mL), free TAPBPR (1.12 mL), and free H2-D b (1.20 mL). ( B) Deconvolution of size-exclusion chromatogram from TAPBPR wt complex formation (experiment independent of the sample shown in ( A)). The different elution volumes of the first main peak, marked by dashed lines, already hint at different complex stabilities. ( A) H2-D b (10 µM) loaded with a photo-cleavable peptide (RGPGRAFJ*TI, J* denotes photocleavable amino acid) was irradiated with UV light in the presence of TAPBPR wt (3 µM, red), TAPBPR Tsn-SL (blue), or TAPBPR ΔSL (yellow) and subsequently analyzed by SEC. Abbreviations: MHC I hc: MHC I heavy chain wt: wildtype Tsn: tapasin SL: scoop loop M: protein marker kDa: kilodalton A 280: absorption at 280 nm V 0: void volume V t: total volume. ( F) The TAPBPR proteins, injected at different concentrations to facilitate comparison, eluted as monodisperse samples during size-exclusion chromatography (SEC). The MHC I allomorphs H2-D b (mouse) and HLA-A*02:01 (human) were refolded in the presence of β2m and peptide. ( D, E) Purified proteins used in the current study were analyzed by non-reducing SDS-PAGE. ( C) Sequence alignment of the scoop-loop region in the TAPBPR constructs used in this study. The viewing direction is indicated by the black arrow in panel ( A). The width of the helix cartoons has been reduced to facilitate visualization of the electron density. ( B) 2F o-F c electron density of the X-ray structure in the region of the scoop loop, contoured at 0.8σ. The zoom-in shows how the TAPBPR scoop loop (purple) is inserted into the F-pocket region of the MHC I peptide-binding groove that is occupied by the C terminus of the peptide before peptide displacement. ( A) X-ray structure of the TAPBPR-MHC I complex in cartoon representation (PDB ID: 5OPI).
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