Fluorescence study of a mutant cytochrome b5 with a single tryptophan in the membrane-binding domain

Abstract

Fluorescence studies of cytochrome b5 are complicated by the presence of three tryptophans, at positions 108, 109, and 112, in the membrane-binding domain. The cDNA for rabbit liver cytochrome b5, isolated from a lambda gt11 library, was used to generate a mutated mRNA where the codons for tryptophans-108 and -112 were replaced by codons for leucine. The sequence was expressed in Escherichia coli and the mutant protein was isolated. This mutant protein had the expected absorption spectrum, and its amino acid composition was confirmed by amino acid analysis and by DNA sequencing of the construct. The fluorescence emission spectrum of the mutant is blue-shifted and is narrower than that of the native protein. The quantum yield of the mutant protein, per molecule, is only 60% of that of the native protein, and the enhancement when bound to lipid vesicles or detergent micelles is higher for the mutant. Fluorescence anisotropy measurements and quenching studies using brominated lipids suggest that the fluorescence of the native protein is due to tryptophans-109 and -108 while tryptophan-112 does not emit but undergoes nonradiative energy transfer to tryptophan-108. With this mutant, it was shown that incomplete energy transfer from tyrosines-126 and -129 to tryptophan-109 occurs when the membrane binding domain is inserted into lipid vesicles, which suggests that the membrane-binding domain does not exist in a tight hairpin loop.

Spectroscopic studies of the interaction of Ca2+‐ATPase‐peptides with dodecyl maltoside and its brominated analog

“…To date, brominated phospholipids have been used to analyze binding of protein to vesicles [37,63], relative affinities of lipids to membrane proteins [64,65] and, through bromination at different positions in the alkyl chain, to investigate the topography of integral membrane proteins (e.g. [35,66]). Brominated detergents [36,38,43,67] appear to be a useful complement to brominated phospholipids to study membrane proteins.…”

Exploring membrane organization and dynamics by the wavelength-selective fluorescence approach

“…REES is indeed observed when the above criteria are satisfied (Ladokhin et al, 1991;Mukherjee, 1993, 1999a,b;Chattopadhyay and Rukmini, 1993;Hutterer et al, 1996;Chattopadhyay et al, 1997;Ghosh et al, 1997;Santos et al, 1998;MacPhee et al, 1999;Raja et al, 1999;Granjon et al, 2001). In addition, it is preferable that the membrane-embedded molecule has only one fluorescent group which has a unique location in the membrane and not a distribution of locations.…”

“…In addition, the red edge effect has also been utilized to study the microconformational heterogeneity of the membrane-binding domain of cytochrome b 5 by comparing the information obtained from the native protein and its mutant which has a single tryptophan residue in this domain (Ladokhin et al, 1991). Both these proteins show a red shift in the emission spectrum when excited at the long wavelength edge of the excitation spectrum, indicating thereby that the tryptophan residue(s) in both cases are localized in a region of motional constraint.…”

Fluorophore environments in membrane-bound probes: A red edge excitation shift study

“…The fluorescence probe TNS shows no such red edge excitation effect in methanol or dioxane (Lakowicz & Keating-Nakamoto, 1984) but shows REES when bound to proteins such as apomyoglobin, &lactoglobulin, &casein, the human a'-acid glycoprotein, and the serum albumins (Demchenko, 1982;Lakowicz & Keating-Nakamoto, 1984;Albani, 1992). REES of the tryptophan fluorescence of the intact eye lens (Rao et al, 1989), and of native and mutant cytochrome bs (Ladokhin et al, 1991), has also been recently reported.…”