Fluorescence Study Of A Temperature-induced Conversion From The Loose To The Tight-binding Form Of Membrane-bound Cytochrome-b5

Fluorescence Study Of A Temperature-induced Conversion From The Loose To The Tight-binding Form Of Membrane-bound Cytochrome-b5

Ladokhin A S; Wang L; Steggles A W; Malak H; Holloway P W

Biochemistry

1993

1993-07

Cytochrome b5 is a liver integral membrane protein that has now been expressed in, and isolated from, Escherichia coli. The structure-function relationships of the 43 amino acid membrane-binding domain (nonpolar peptide) have been examined in both native and mutant forms of the protein; in the latter, tryptophan residues at positions 108 and 112 were replaced by leucine. The temperature dependence of the fluorescence quantum yield of the Trp residues in the isolated membrane-binding domain was examined while the domain was bound to lipid vesicles. Both the lipid-bound mutant domain and lipid-bound native domain showed an irreversible increase in fluorescence above 50-degrees-C. When the whole cytochrome b5 molecule, bound to lipid vesicles, was heated to this temperature, there was a conversion of the metastable, intermembrane-exchangeable (''loosely'' bound), conformation to a final, virtually unexchangeable (''tightly'' bound), conformation. It has been suggested previously that the protein exists in a ''looped back'' conformation and a ''bilayer penetrating'' conformation. Although the present studies are not designed to determine the absolute conformations of the loose and tight forms, the changes observed in steady-state and frequency-modulated fluorescence and the lack of change in depth of Trp 109 in the bilayer are consistent with a movement of the C-terminal segment from a looped back to a bilayer penetrating conformation as the tight form is generated.

bilayers; Biochemistry & Molecular Biology; cross-linking; decay; domain; embedded segment; orientation; photoactivatable phospholipids; topography; vesicles

Journal Article or Conference Abstract Publication

Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).

6951-6956

27

32