Determination of the topography of cytochrome b5 in lipid vesicles by fluorescence quenching

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Abstract
Cytochrome b5, a protein isolated from the endoplasmic reticulum by detergent extraction, interacts spontaneously with small unilamellar phosphatidylcholine vesicles. When the vesicles are made from 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), the tryptophan fluorescence of the cytochrome is enhanced, and when they are made from 1-palmitoyl-2-(dibromostearoyl)phosphatidylcholine (BRPC), the fluorescence is quenched. A series of BRPC were synthesized with Br atoms at the 6,7, 9,10, 11,12 or 15,16 positions. The vesicles synthesized from each of these lipids were similar in size to those made from POPC. The relative fluorescence intensities of the cytochrome b5 in POPC and 6,7-, 9,10-, 11,12- and 15,16-BRPC were 100, 19.4, 29.4, 37.1 and 54.0, respectively. These data suggest that the exposed tryptophan(s) is (are) at a depth of 0.7 nm below the surface of the vesicle. Br is a collisional quencher; hence, these data may indicate the relative position of the lipid annulus around the protein rather than the depth of the protein below the average vesicle surface. Cytochrome b5 contains 3 potentially fluorescent tryptophans, and determinations of fluorescent quantum yield indicate all 3 are fluorescent with an average quantum yield, when in POPC vesicles, of 0.21. Fluorescence lifetime measurements by the demodulation technique indicated heterogeneity of fluorescence lifetimes in all vesicles. The lifetimes in the BRPC vesicles ranged from 2.0-2.4 ns compared to a value of 3.3 ns in POPC. Quenching of fluorescence in vesicles composed of mixtures of POPC and a BRPC indicated that the quenchable tryptophan(s) was (were) well shielded from the bromo lipid with perhaps only a 75.degree. angle of approach. This study suggests that specifically brominated lipid can be used to determine the depth and exposure of tryptophans in membrane binding domains of proteins.