Thermostability analysis of major histocompatibility complex class I molecules by temperature gradient gel...

Empty major histocompatibility complex (MHC) class I molecules present on the surface of RMA-S (26 degrees C) cells were loaded with the iodinated peptides APGNYPAL, FAPGNYPAL (SEV-9) and RGYVYQGL (VSV-8), respectively. The thermostability of these peptide-loaded MHC class I molecules was assessed using temperature gradient native polyacrylamide gel 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 signal due to released peptide. APGNYPAL-loaded class I molecules melted at 11 degrees C with considerable release even at 0 degrees C. VSV-8-loaded class I molecules melted first at 36 degrees C, whereas SEV-9-loaded molecules melted at about 22 degrees C. A discrimination between the binding of SEV-9 to Kb and Db molecules was seen in the melting patterns. Results are discussed in correlation with known crystallographic structures of class I molecules containing peptides in the binding groove.