Fig. 3: Capturing empty HLA-A*02:01/TAPBPR complexes for peptide exchange.
From: High throughput pMHC-I tetramer library production using chaperone-mediated peptide exchange
a Structure-based design of goldilocks peptides: comparison of polar contacts between HLA-A*02:01 and the N-terminal region of LLFGYPVYV (TAX) peptide (upper left). _LFGYPVPYV (gTAX) (upper right), Ac-LLFGYPVYV (bottom right) and lLFGYPVYV where l = D-Leucine (bottom left). Structures were modeled using PDB ID IDUZ26. b Peptide complex thermal stabilities of HLA-A*02:01 bound to TAX, lLFGYPVYV, Ac-LLFGYPVYV and gTAX. c SEC TAPBPR binding assays of TAX/HLA-A02:01 (left), gTAX/HLA-A02:01 (right). d Native gel electrophoresis of HLA-A*02:01/TAPBPR complex incubated with a 10-fold molar excess of a non-specific peptide (p29, YPNVNIHNF) or varying molar excess of a specific, high-affinity peptide (TAX). 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. e Competitive binding of TAMRA-TAX to purified HLA-A*02:01/TAPBPR complexes from (c) as a function of increasing peptide concentration, measured by fluorescence polarization. f Conceptual diagram of TAPBPR-mediated capture and peptide loading on empty MHC-I molecules. g Bio-Layer Interferometry analysis of TAPBPR dissociation from HLA-A*02:01 in the presence of peptides of different affinities. Data shown in e and g are representative of triplicate assays (n = 3) and error-bars are standard deviations from the mean. Source data are provided as a Source Data file.
