Fluorescence study of a mutant cytochrome b5 with a single tryptophan in the membrane-binding domain.
Fluorescence studies of cytochrome b5 are complicated by the presence of three tryptophans, at positions 108, 109, and 112, in the membrane-binding domain. The cDNA for rabbit liver cytochrome b5, isolated from a lambda gt11 library, was used to generate a mutated mRNA where the codons for tryptophans-108 and -112 were replaced by codons for leucine. The sequence was expressed in Escherichia coli and the mutant protein was isolated. This mutant protein had the expected absorption spectrum, and its amino acid composition was confirmed by amino acid analysis and by DNA sequencing of the construct. The fluorescence emission spectrum of the mutant is blue-shifted and is narrower than that of the native protein. The quantum yield of the mutant protein, per molecule, is only 60% of that of the native protein, and the enhancement when bound to lipid vesicles or detergent micelles is higher for the mutant. Fluorescence anisotropy measurements and quenching studies using brominated lipids suggest that the fluorescence of the native protein is due to tryptophans-109 and -108 while tryptophan-112 does not emit but undergoes nonradiative energy transfer to tryptophan-108. With this mutant, it was shown that incomplete energy transfer from tyrosines-126 and -129 to tryptophan-109 occurs when the membrane binding domain is inserted into lipid vesicles, which suggests that the membrane-binding domain does not exist in a tight hairpin loop.
Department of Biochemistry, University of Virginia Health Sciences Center, Charlottesville 22908.
A S
AS
Ladokhin
L
L
Wang
A W
AW
Steggles
P W
PW
Holloway
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002462:Cell Membrane / Q000737:chemistry; D015786:Cytochromes b5 / Q000737:chemistry / Q000235:genetics*; D008055:Lipids / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D009154:Mutation; D017830:Octoxynol; D011092:Polyethylene Glycols; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000235:genetics*
D008055:Lipids; D008565:Membrane Proteins; D011092:Polyethylene Glycols; D014364:Tryptophan; D017830:Octoxynol; D015786:Cytochromes b5