Amino acid substitutions in the membrane-binding domain of cytochrome b5 alter its membrane-binding properties.
The structure-function relationships of the 43-amino-acid membrane-binding domain of cytochrome b5 have been examined in two mutant forms of the protein. In one mutant, two tryptophans in the membrane-binding domain, at positions 108 and 112, were replaced by leucines, and in the second mutant, in addition, aspartic acid 103 was also replaced by leucine. The fluorescence emission spectra of the three proteins and their degree of quenching by brominated lipids indicate that the mutations are not producing major conformational changes or allowing a deeper degree of penetration of the domain into the bilayer. The hydrophobicities of the three proteins were compared, by determining strengths of self-association and membrane affinities, and it was found that the protein with two additional leucines was much less hydrophobic and the one with three additional leucines was much more hydrophobic than the native cytochrome. It appears that small changes in amino acid composition, which produce no gross changes in the structure of the membrane-binding domain, will nevertheless produce very large changes in the strengths of self- and membrane-association. These differences in self-association had profound effects on the times required for membrane-association to reach equilibrium.
Tretyachenko-Ladokhina
V G
VG
Department of Biochemistry, University of Virginia Health Sciences Center, Charlottesville 22908.
Ladokhin
A S
AS
Wang
L
L
Steggles
A W
AW
Holloway
P W
PW
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002850:Chromatography, Gel; D015786:Cytochromes b5 / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D008081:Liposomes; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D008081:Liposomes; D011994:Recombinant Proteins; D015786:Cytochromes b5