Effect of lipid unsaturation on the binding of native and a mutant form of cytochrome b5 to membranes.
The partitioning of native cytochrome b5 and a mutant form, where Trp-108 and Trp-112 were both replaced by Leu, into small unilamellar lipid vesicles was examined. The vesicles were made from phosphatidylcholines containing mono- and di-unsaturated acyl chains. As these amphipathic proteins self-associate in aqueous solution, the binding was not monitored by a simple lipid titration experiment but by an exchange assay using fluorescence quenching by brominated lipids. Each protein had a greater affinity for lipids containing mono-unsaturated chains than for vesicles containing di-unsaturated chains, and the affinities of both proteins increased in buffers of higher ionic strength. The native protein had a higher affinity than the mutant protein for all vesicles; the ratio of the affinities was relatively constant at approximately 30. This corresponds to a difference in the free energy of partitioning of 2 kcal mol(-)(1). The fluorescence quantum yields of both proteins were much lower in lipids with di-unsaturated chains whereas a similar lowering was not seen with a simple Trp compound. These data suggest that the decreased membrane hydrophobicity seen by the proteins in di-unsaturated membranes is not an inherent property of the bilayer but is induced by the insertion of the protein. Further, the similar behavior of the two proteins suggests this modulation is not sensitive to the amino acid side chains of the inserted domain.
Başaran
N
N
TUBITAK, Türkiye Bilimsel Ve Teknik Araştirma Kurumu, Atatürk, Ankara, Turkey.
Doebler
R W
RW
Goldston
H
H
Holloway
P W
PW
D001966:Bromine / Q000737:chemistry; D002850:Chromatography, Gel; D015786:Cytochromes b5 / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009994:Osmolar Concentration; D010713:Phosphatidylcholines / Q000737:chemistry; D011485:Protein Binding / Q000235:genetics; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000031:analogs & derivatives / Q000737:chemistry
D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D008565:Membrane Proteins; D010713:Phosphatidylcholines; C097263:tryptophan octyl ester; C060612:1-stearoyl-2-linoleoylphosphatidylcholine; D014364:Tryptophan; D015786:Cytochromes b5; C017251:1,2-oleoylphosphatidylcholine; D001966:Bromine; C028694:1-palmitoyl-2-oleoylphosphatidylcholine