Fluorescence study of a temperature-induced conversion from the "loose" to the "tight" binding form of membrane-bound cytochrome b5

Biochemistry. 1993 Jul 13;32(27):6951-6. doi: 10.1021/bi00078a020.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Cell Membrane / enzymology
  • Cytochromes b5 / chemistry
  • Cytochromes b5 / metabolism*
  • Fluorescence Polarization
  • Liver / enzymology
  • Membrane Proteins / metabolism
  • Phosphatidylcholines / metabolism
  • Protein Binding
  • Rabbits
  • Temperature


  • Membrane Proteins
  • Phosphatidylcholines
  • Cytochromes b5