Fig. 4: TAPBPR-mediated peptide exchange on HLA-A*24:02 and HLA-A*68:02.
From: High throughput pMHC-I tetramer library production using chaperone-mediated peptide exchange
a Structure-based design of goldilocks peptides: analysis of A-pocket hydrogen bonds observed in the X-ray structure of HLA-A*24:02/Nef134-10 (RYPLTFGWCF) and homology-based model of HLA-A*28:02/TAX (LLFGYPVYV). Peptide residues are labelled in red, A-pocket MHC residues in black. The structure of HLA-A*24:02/Nef134-10 was obtained from PDB ID 3QZW41. The structure of HLA-A*68:02/TAX was modeled using a related structure (PDB ID 4HX142) as input. b Native gel electrophoresis analysis showing MHC-I/TAPBPR complex dissociation in the presence of 10-fold molar excess of relevant, high-affinity peptides PHOX2B (QYNPIRTTF), Nef134-10 (lanes 5 and 6) and TAX, MART-1 (lanes 10 and 11) for complexes prepared using refolded HLA-A*24:02 and HLA-A*68:02 with gNEF (lane 2) and gTAX (lane 7) goldilocks peptides, respectively (protein yields of approximately 6 and 8 mg from a 1 L refolding reaction). The exchanged pMHC molecular species on lanes 5,6,10 and 11 are indicated with arrows, showing electrophoretic mobilities that depend on the charge of the bound peptide. Both complexes remain bound in the presence of 10-fold molar excess of the irrelevant peptide p29 (YPNVNIHNF) (lanes 4 and 9). 12% polyacrylamide native gels were run at 90 V for 5 h at 4 °C before visualization with InstantBlue (Expedeon). Data shown are representative of triplicate gel assays. All protein samples used in a and b were derived from the same peptide exchange experiment, and the gels were processed in parallel. c Overlaid Differential Scanning Fluorimetry temperature profiles of: goldilocks/MHC-I (red) and high-affinity peptide/MHC-I (blue) prepared using chaperone-mediated exchange of the goldilocks for high-affinity peptides, followed by purification of the pMHC peak by SEC. Thermal stabilities are shown: HLA-A*24:02 refolded with gNEF (YPLTFGWCF) or exchanged with NEF (RYPLTFGWCF) (left), and HLA-A*68:02 refolded with gTAX (_LFGYPVYV) or exchanged with TAX (LLFGYPVYV) (right). The peaks at approximately 63 °C correspond to the thermal melt of the β2m light chain37. Data shown are representative of triplicate assays and error-bars are standard deviation from the mean. Source data are provided as a Source Data file.
