Peptide-MHC I complex stability measured by nanoscale differential scanning fluorimetry reveals molecular mechanism of thermal denaturation.

Recombinant major histocompatibility complex class I molecules are used in diagnostic and therapeutic approaches in cancer immunotherapy, with many studies exploring their binding to antigenic peptides. Current techniques for kinetic peptide binding studies are hampered by high sample consumption, low throughput, interference with protein stability, and/or high background signal. Here, we validate nanoscale differential scanning fluorimetry (nanoDSF), a method using the tryptophan fluorescence of class I molecules, for class I/peptide binding, and we use it to determine the molecular mechanism of the thermal denaturation of HLA-A*02:01.