The effects of phospholipid unsaturation and alcohol perturbation at the protein/lipid interface probed using fluorophore lifetime heterogeneity.
The influence of phospholipid unsaturation and perturbation by alcohols, on the membrane protein/lipid interface, was probed using the fluorescence decay properties of 1,6-diphenyl-1,3,5-hexatriene (DPH) and DPH attached to the sn-2 chain of phosphatidylcholine (DPH-PC), in lipid bilayers and microsomal membranes. With microsomal membranes it was found that it was appropriate to describe the fluorescence decay of DPH-PC as a range of decay rates, accomplished by fitting the data to a bimodal fluorescence lifetime distribution. The major lifetime center had a broad distributional width, indicative of excited state fluorophore heterogeneity. The effect was attributable to protein, and by inference, the protein/lipid interface, since in vesicles made from total microsomal lipids (i.e., without protein) the fluorescence decay was homogeneous. Upon addition of ethanol or hexanol the width of the lifetime distribution of the major lifetime center increased, indicating increased environmental heterogeneity. It was confirmed that the effect was manifest at the protein/lipid interface, and not due to lipid-reorganizational factors, since it could also be obtained using a simple lipid bilayer vesicle system with apocytochrome c as a model membrane protein, and DPH instead of DPH-PC. Environmental heterogeneity was also found to increase with increased phosphatidylcholine (sn-2) unsaturation. The environmental heterogeneity at the protein/lipid interface could arise from a combination of varying polarities of amino acid side chains and of water that may intercalate in packing defects on the hydrophobic surface of the protein. Therefore the results could be explained on the basis of an increased degree of hydration at the protein/lipid interface. Such an effect offers a route whereby acyl chain perturbation and increased unsaturation might influence protein conformation and hence function.
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Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107.
Kelly
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Stubbs
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D000438:Alcohols / Q000737:chemistry*; D000818:Animals; D001059:Apoproteins / Q000737:chemistry*; D003574:Cytochrome c Group / Q000737:chemistry*; D045304:Cytochromes c; D005456:Fluorescent Dyes; D007425:Intracellular Membranes / Q000737:chemistry; D010713:Phosphatidylcholines; D010743:Phospholipids / Q000737:chemistry*; D051381:Rats
D000438:Alcohols; D001059:Apoproteins; D003574:Cytochrome c Group; D005456:Fluorescent Dyes; D010713:Phosphatidylcholines; D010743:Phospholipids; C060859:2-(3-(diphenylhexatrienyl)propanoyl)-3-palmitoylphosphatidylcholine; D045304:Cytochromes c