Calorimetric and Fluorescence Characterization of Interactions between Cytochrome b5 and Phosphatidylcholine Bilayers

Ernesto Freire, Tom Markello, Christopher Rigell, Peter W. Holloway

Research output: Contribution to journalArticlepeer-review

Abstract

The interactions of cytochrome b5 with dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine lipid bilayers have been studied with high-sensitivity differential scanning calorimetry and fluorescence spectroscopy. The incorporation of cytochrome b5 into large single lamellar vesicles causes a reduction in the enthalpy change associated with the lipid phase transition. Analysis of the dependence of this enthalpy change on the protein/lipid molar ratio indicates that each cytochrome b5 molecule prevents 14 ° 1 lipid molecules from participating in the gel to liquid-crystalline transition and that this number is independent of the phospholipid acyl chain length. Resonance energy transfer between the intrinsic tryptophan fluorescence of cytochrome b5 and pyrenedecanoic acid indicates that, in the liquid-crystalline phase, protein and lipid molecules are uniformly distributed within the bilayer plane. In the gel phase, pyrenedecanoic acid partitions into the boundary layer lipid causing a dramatic decrease in the fluorescence intensity of cytochrome b5 The excimer/monomer ratios of pyrenedecanoic acid decrease upon increasing the protein/lipid molar ratio, indicating that the presence of protein molecules within the bilayer slows down the lateral mobility of the lipid probes. The picture that emerges from this set of experiments is that cytochrome b5 perturbs one layer of lipid around the hydrophobic segment of the protein and that this layer is unable to undergo the gel-liquid-crystalline transition, remaining instead in a relatively disordered configuration above and below the transition temperature of the bulk lipid.

Original languageEnglish (US)
Pages (from-to)1675-1680
Number of pages6
JournalBiochemistry
Volume22
Issue number7
DOIs
StatePublished - 1983

ASJC Scopus subject areas

  • Biochemistry

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