Extension of the parallax analysis of membrane penetration depth to the polar region of model membranes: Use of fluorescence quenching by a spin-label attached to the phospholipid polar headgroup

Abstract

The parallax method is a method by which the depth of fluorescent molecules within a membrane is calculated from the ratio of quenching induced by two spin-labeled phospholipids at different depths. In this report, the method is extended to measurements of depth in the polar headgroup region of the membrane through use of a lipid with a spin-label attached to the polar choline moiety. Quenching data indicate that the choline-attached nitroxide is close to 19.5 A from the bilayer center, in good agreement with the choline location previously determined by diffraction measurements. By using quenching results obtained with this polar headgroup-labeled phospholipid, depths more accurate than those measured previously can be obtained for fluorophores in the polar region of the membrane. It appears that the most reliable results are obtained when depth is calculated from the quenching of the two spin-labels that quench a specific fluorophore most strongly. Applying this approach to a series of anthroyloxy-labeled fatty acids indicates that the depth of the anthroyloxy group is almost linearly related to the number of carbon atoms between it and the carboxyl group. The fatty acid carboxyl group itself is close to 18.6 A from the bilayer center in the ionized form and 16 A from bilayer center in the protonated form. This is close to the depth of the carboxyl groups on phospholipid fatty acyl chains. More accurate depths have also been obtained for 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD) labeled phospholipids using the quenching of the choline-attached spin-label.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular rheology of neuronal membranes explored using a molecular rotor: Implications for receptor function

“…The actual spin (nitroxide) contents of the spin-labeled phospholipids (Tempo-, 5-and 12-PC) were assayed using fluorescence quenching of anthroyloxy-labeled fatty acids (2-and 12-AS) as described previously (Abrams and London, 1993). For depth measurements, unilamellar vesicles (ULV) of DOPC containing 10% (mol/mol) spin-labeled phospholipids (Tempo-, 5-or 12-PC) labeled with 1% (mol/mol) of BODIPY-C12 were prepared by the ethanol injection method (Kremer et al, 1977).…”

Quantification of protein–lipid selectivity using FRET

“…Using a binomial distribution the probability of each occupation number (0–12 sites occupied simultaneously by labeled lipid) is calculated, and the FRET contribution arising from energy transfer to annular lipids is given bywhere k T is the energy transfer rate for an acceptor located in an annular site:τ D is the donor lifetime in the absence of acceptor, and d is the distance from the donor (in the protein) to the acceptor inside the annular lipid shell. The value of d was calculated from published data on the position of the acceptor fluorophores (NBD) in the labeled phospholipids incorporated in lipid bilayers (Abrams and London 1993; Màzeres et al. 1996).…”

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

“…The precise orientation and location of the NBD group of this molecule in the membrane is known London, 1987, 1988;Mitra and Hammes, 1990;Wolf et al, 1992;Abrams and London, 1993). This group has been found to be localized at the membrane interface which has unique motional and dielectric characteristics distinct from both the bulk aqueous phase and the hydrocarbon-like interior of the membrane (Seelig, 1977;Ashcroft et al, 1981;Stubbs et al, 1985;Perochon et al, 1992;Slater et al, 1993;Venable et al, 1993;White and Wimley, 1994;Gawrisch et al, 1995) thus making it an ideal probe for monitoring red edge effects.…”