A. Tulp et Dj. Verwoerd, THERMOSTABILITY ANALYSIS OF MAJOR HISTOCOMPATIBILITY COMPLEX CLASS-I MOLECULES BY TEMPERATURE-GRADIENT GEL-ELECTROPHORESIS, European Journal of Immunology, 23(8), 1993, pp. 2030-2033
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.