Fluorescence study of a temperature-induced conversion from the "loose" to the "tight" binding form of membrane-bound cytochrome b5.
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.
Ladokhin
A S
AS
Department of Biochemistry, University of Virginia Health Sciences Center, Charlottesville 22908.
Wang
L
L
Steggles
A W
AW
Malak
H
H
Holloway
P W
PW
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D015786:Cytochromes b5 / Q000737:chemistry / Q000378:metabolism*; D005454:Fluorescence Polarization; D008099:Liver / Q000201:enzymology; D008565:Membrane Proteins / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D013696:Temperature
D008565:Membrane Proteins; D010713:Phosphatidylcholines; D015786:Cytochromes b5