Determination of the transbilayer distribution of fluorescent lipid analogs by nonradiative fluorescence resonance energy transfer

Abstract

We measured the nonradiative fluorescence resonance energy transfer between 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD) labeled lipids (amine labeled phosphatidylethanolamine or acyl chain labeled phosphatidylcholine) and rhodamine labeled lipids in large unilamellar dioleoylphosphatidylcholine vesicles. Two new rhodamine labeled lipid analogues, one a derivative of monolauroylphosphatidylethanolamine and the other of sphingosylphosphorylcholine, were found to exchange through the aqueous phase between vesicle populations but not to be capable of rapid transbilayer movement between leaflets. Energy transfer from NBD to rhodamine was measured using liposomes with symmetric or asymmetric distributions of these new rhodamine labeled lipid analogues to determine the relative contributions of energy transfer between donor and acceptor fluorophores in the same (cis) and opposite (trans) leaflets. Since the characteristic R0 values for energy transfer ranged from 47 to 73 A in all cases, significant contributions from both cis and trans energy transfer were observed. Therefore, neither of these probes acts strictly as a half-bilayer quencher of NBD lipid fluorescence. The dependence of transfer efficiency on acceptor density was fitted to a theoretical treatment of energy transfer to determine the distances of closest approach for cis and trans transfer. These parameters set limits on the positions of the fluorescent groups relative to the bilayer center, 20-31 A for NBD and 31-55 A for rhodamine, and provide a basis for future use of these analogues in measurements of transbilayer distribution and transport.

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.…”

“…NBD-PE exhibits REES in model membranes of DOPC Mukherjee, 1993, 1999b). Since the precise localization of the fluorescent NBD group in membranebound NBD-PE is known to be interfacial London, 1987, 1988;Mitra and Hammes, 1990;Wolf et al, 1992;Abrams and London, 1993), this result directly implies that the interfacial region of the membrane offers considerable restriction to the reorientational motion of the solvent dipoles around the excited state fluorophore. In another study, REES of membrane-bound NBDÁ/cholesterol, in which the NBD group is covalently attached to the flexible chain of the cholesterol molecule, was reported (Chattopadhyay and Mukherjee, 1999b).…”

γ-Cyclodextrins Greatly Enhance Translocation of Hydrophobic Fluorescent Phospholipids from Vesicles to Cells in Culture

“…In this assay, the donor vesicles contain the pyrene lipid under study, TNP-PE, a quencher of pyrene fluorescence, and unlabeled phospholipids, whereas the acceptor vesicles consist of unlabeled lipids only. Transfer of the pyrene lipid molecules from the donor to the acceptor vesicles results in dequenching 1 The abbreviations used are: PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; SM, sphingomyelin; Bodipy, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-S-indacene; Bodipy 12 , Bodipy-3-dodecanoylate; Bodipy 12 -PC, 1-hexadecanoyl,2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl)-snglycero-3-PC; Bodipy 12 -PE, 1-hexadecanoyl,2-(4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-dodecanoyl)-sn-glycero-3-PE; Bodipy 12 -SM, n- (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl)-SM; BP, band pass; CE-␥-CD, carboxyethyl-␥-cyclodextrin; NBD 6 PC, 2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl-sn-glycero-3-PC; NBD 12 PS, 2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3-PS; POPA, palmitoyl-oleoyl-phosphatidic acid; POPC, palmitoyl-oleoyl-phosphatidylcholine; Pyr n PC, 16:0-pyrenylacyl-PC (n ϭ carbons in the acyl chain); DiPyr n PC, dipyrenylacyl-PC; TNP-PE, N-trinitrophenyl-PE; I-MEM, CO 2 -independent medium, Life Technologies catalog number 18045; HPLC, high pressure liquid chromatography. 2 Tanhuanpää , Virtanen, J.…”

“…One reason for this is that short chain NBD 6 -phospholipids are rapidly degraded (half-life is 1 h or less) in mammalian cells at the physiological temperature (8 -10). The altered conformation of such lipids (11,12) may render them targets for some cellular phospholipases. However, an even more serious limitation is that the short chain lipids, unlike the natural ones, can distribute spontaneously between all accessible membrane surfaces due to their hydrophilicity (8).…”

Micellar Organization and Dynamics: A Wavelength-Selective Fluorescence Approach

“…43,44 In NBD-PE, the NBD group is covalently attached to the headgroup of a phosphatidylethanolamine molecule. The NBD group in NBD-PE has earlier been shown to be localized in the interfacial region of the membrane [45][46][47][48][49][50] and its location in the micellar environment is most likely to be interfacial. It is thus of interest to investigate wavelength-selective fluorescence effects in micellar assemblies, especially since micelles are more dynamic structures than membranes and probably have more water in their core.…”