THERMOSTABILITY ANALYSIS OF MAJOR HISTOCOMPATIBILITY COMPLEX CLASS-I MOLECULES BY TEMPERATURE-GRADIENT GEL-ELECTROPHORESIS

Empty major histocompatibility complex (MHC) class I molecules present on the surface of RMA-S (26-degrees-C) cells were loaded with the iod inated peptides APGNYPAL, FAPGNYPAL (SEV-9) and RGYVYQGL (VSV-8), resp ectively. The thermostability of these peptide-loaded MHC class I mole cules was assessed using temperature gradient native polyacrylamide ge l electrophoresis. A linear temperature gradient perpendicular to the direction of electrophoresis yielded a graphical representation of the melting of MHC class I molecules. The class I signal disappeared when the peptide melted out of the groove, and gave rise to a second signa l due to released peptide. APGNYPAL-loaded class I molecules melted at 11-degrees-C with considerable release even at 0-degrees-C. VSV-8-loa ded class I molecules melted first at 36-degrees-C, whereas SEV-9-load ed molecules melted at about 22-degrees-C. A discrimination between th e binding of SEV-9 to K(b) and D(b) molecules was seen in the melting patterns. Results are discussed in correlation with known crystallogra phic structures of class I molecules containing peptides in the bindin g groove.