Effect of lipid unsaturation on the binding of native and a mutant form ofcytochrome b(5) to membranes

The partitioning of native cytochrome bs and a mutant form, where Trp-108 a nd 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-as sociate in aqueous solution, the binding was not monitored by a simple lipi d titration experiment but by an exchange assay using fluorescence quenchin g by brominated lipids. Each protein had a greater affinity for lipids cont aining 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 pr otein for all vesicles; the ratio of the affinities was relatively constant at approximately 30, This corresponds to a difference in the free energy o f partitioning of 2 kcal mol(-1) The fluorescence quantum yields of both pr oteins were much lower in lipids with di-unsaturated chains whereas a simil ar lowering was not seen with a simple Trp compound. These data suggest tha t the decreased membrane hydrophobicity seen by the proteins in di-unsatura ted 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 prot eins suggests this modulation is not sensitive to the amino acid side chain s of the inserted domain.