FLUORESCENCE STUDY OF A TEMPERATURE-INDUCED CONVERSION FROM THE LOOSETO THE TIGHT-BINDING FORM OF MEMBRANE-BOUND CYTOCHROME-B5

Cytochrome b5 is a liver integral membrane protein that has now been e xpressed in, and isolated from, Escherichia coli. The structure-functi on relationships of the 43 amino acid membrane-binding domain (nonpola r peptide) have been examined in both native and mutant forms of the p rotein; in the latter, tryptophan residues at positions 108 and 112 we re replaced by leucine. The temperature dependence of the fluorescence quantum yield of the Trp residues in the isolated membrane-binding do main was examined while the domain was bound to lipid vesicles. Both t he lipid-bound mutant domain and lipid-bound native domain showed an i rreversible increase in fluorescence above 50-degrees-C. When the whol e cytochrome b5 molecule, bound to lipid vesicles, was heated to this temperature, there was a conversion of the metastable, intermembrane-e xchangeable (''loosely'' bound), conformation to a final, virtually un exchangeable (''tightly'' bound), conformation. It has been suggested previously that the protein exists in a ''looped back'' conformation a nd a ''bilayer penetrating'' conformation. Although the present studie s are not designed to determine the absolute conformations of the loos e and tight forms, the changes observed in steady-state and frequency- modulated fluorescence and the lack of change in depth of Trp 109 in t he bilayer are consistent with a movement of the C-terminal segment fr om a looped back to a bilayer penetrating conformation as the tight fo rm is generated.