Determination of the topography of cytochrome b5 in lipid vesicles by fluorescence quenching.
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 bromine 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. Bromine 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 three potentially fluorescent tryptophans, and determinations of fluorescent quantum yield indicate all three potentially fluorescent tryptophans, and determinations of fluorescent quantum yield indicate all three 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 to 2.4 ns compared to a value of 3.3 ns in POPC.(ABSTRACT TRUNCATED AT 250 WORDS)
D002152:Calorimetry, Differential Scanning; D003573:Cytochrome b Group / Q000378:metabolism*; D015786:Cytochromes b5; D007700:Kinetics; D008081:Liposomes; D010713:Phosphatidylcholines / Q000138:chemical synthesis; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence / Q000379:methods; D013329:Structure-Activity Relationship
D003573:Cytochrome b Group; D008081:Liposomes; D010713:Phosphatidylcholines; D015786:Cytochromes b5; C028694:1-palmitoyl-2-oleoylphosphatidylcholine