Binding and fluorescence properties of the membrane domain of NADH-cytochrome-b5 reductase: determination of the depth of Trp-16 in the bilayer

Abstract The membrane domain of NADH-cytochrome-b5 reductase, which extends from the amino-terminal myristic acid through the first 28 amino acid residues, can be isolated in cholate after a mild trypsin treatment of cholate-solubilized reductase, and in phospholipid vesicles after exhaustive trypsin treatment of vesicle-bound reductase. The detergent-solubilized peptide has a high affinity for phospholipid vesicles and can be reconstituted in vesicles by the detergent-dialysis method. The fluorescence of Trp-16 of this peptide is highly sensitive to the polarity of the microenvironment. The fluorescence quantum yield of this residue is 0.10 when the peptide is dispersed in 1% sodium cholate, but 0.46-0.52 when the peptide is reconstituted in phospholipid vesicles. Fluorescence energy transfer from this tryptophan residue in vesicle-bound peptide to a random array of acceptors in the head-group region of the vesicle outer monolayer shows that Trp-16 resides at a depth of 20-23 A in the bilayer.