Fig. 1.
TAPBPR binds HLA-A2 loaded with photosensitive FluM1 peptide (photo-FluM158–66) after UV irradiation. (A) Native gel shift analysis. Individual components: tapasin (lanes 1 and 2), HLA-A2 (lanes 5 and 6), TAPBPR (lanes 7 and 8), and mixtures of tapasin and HLA-A2 (lanes 3 and 4) or TAPBPR and HLA-A2 (lanes 9 and 10) were separated on native gel with (lanes 2, 4, 6, 8, and 10) or without (lanes 1, 3, 5, 7, and 9) UV irradiation as described in Materials and Methods (4 µg of each protein in 10 µL). The positions of migration of the individual components are indicated, and the shifted TAPBPR/HLA-A2 complex band is denoted with a line. (Lanes 1–4 and 5–10, respectively, were taken from different gels run in parallel.) (B) SEC analysis. Individual components, TAPBPR (green), HLA-A2/photo-FluM158–66 (magenta), and the mixture of TAPBPR and HLA-A2 after UV irradiation (blue) were analyzed by SEC (S200 HR10/300) as described in Materials and Methods. (Markers of 158, 44, and 17 kDa eluted at 16.2, 20.1, and 22.6 min, respectively). (C) SDS/PAGE analysis confirms the identity of the TAPBPR/HLA-A2 complex peak. The SEC peaks of TAPBPR/HLA-A2 complex (I) and HLA-A2 (II) were collected, concentrated, and analyzed by SDS/PAGE (5 µg loaded in 10 µL per lane). The bands of TAPBPR, HLA-A2 heavy chain (HLA-A2 HC), and β2m, as well as the position of comigrated molecular-weight standards are indicated. (D) SEC (S200 HR10/300) analysis of tapasin alone (green), HLA-A2/photo-FluM158–66 (magenta), and mixture of tapasin and HLA-A2 following irradiation (blue) shows no complex formation. (E) AUC analysis of TAPBPR (green), HLA-A2/photo-FluM158–66 (blue), and the purified TAPBPR/HLA-A2 complex after UV irradiation (red). Shown are representative normalized sedimentation coefficient distributions at 2 µM. (F) AUC analysis of tapasin (green), HLA-A2/photo-FluM158–66 (blue), and the mixture following irradiation (red). These analyses were performed with 20 µM of each component. (G) AUC analysis of TAPBPR association with HLA-A2/photo-FluM1 without UV irradiation at the representative concentrations indicated. The Inset shows the weighted-average (black) and reaction boundary sedimentation coefficients (gray) for all of the concentrations examined. (Representative sedimentation profiles are shown in Fig. S1.)