Fluorescence energy transfer from diphenylhexatriene to bacteriorhodopsin in lipid vesicles (original) (raw)
Related papers
Biochemistry, 1985
The effect of monomeric bacteriorhodopsin on the lipid order and dynamics in dimyristoylphosphatidylcholine (DMPC) vesicles was monitored as a function of the protein to lipid ratio by timedependent fluorescence anisotropy measurements with diphenylhexatriene (DPH). Energy transfer from the donor DPH to the acceptor retinal of bacteriorhodopsin was used as a spectroscopic ruler to estimate the range of the protein-induced perturbation of the lipid phase. The Forster distance for this donor-acceptor pair is approximately 45 A. Since the effective radius of bacteriorhodopsin is about 17 A, the labels within a neighborhood of radius R , around bacteriorhodopsin are strongly quenched and make a negligible contribution to the end value of the fluorescence anisotropy, from which the order parameter is calculated. Instead, the order parameter is mainly determined by the labels which are more than the Forster distance away from the retinal and which are consequently in the bulk lipid phase. The observed linear increase in order parameter from 0.29 for pure DMPC to 0.62 for a molar bacteriorhodopsin to DMPC ratio of 1/52 thus indicates that the order of the bulk lipids is increased by the interaction with bacteriorhodopsin and that the range of this perturbation is larger than 45 A. In the absence of the acceptor retinal, no energy transfer occurs, and both bulk and boundary lipids are weighted equally in the determination of the order parameter. Only a very small change in the order parameter is observed upon removal of the acceptor, suggesting that bacteriorhodopsin affects the order of all the lipids in roughly the same way. The rotational diffusion constant of DPH determined from the initial slope of the anisotropy decay is independent of the surface concentration of bacteriorhodopsin and of the presence of the acceptor retinal. The viscosity calculated from the rotational diffusion constant is approximately 0.1 P at 35 O C and is an order of magnitude smaller than that determined previously froii the rotational diffusion of bacteriorhodopsin. A comparison of the viscosities determined from the steac pstate and time-resolved fluorescence anisotropy of DPH shows that the first method overestimates the viscosity by as much as a factor of 10.
Journal of Photochemistry and Photobiology A: Chemistry, 1995
Fluorescence energy transfer in lipid vesicles between N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl)-labelled phosphatidylethanolamine (acting as donor) and N-(lissamine-rhodamine B)-labelled phosphatidylethanolamine (acting as acceptor) was studied by steady state and time-resolved fluorescence quenching analysis. Both fluorescent phospholipids were incorporated as minor components in four different types of lipid vesicle: dipalmitoylphosphatidylglycerol vesicles in their L~ gel phase at 20 °C and in their La liquid crystalline phase at 50 °C, and egg yolk phosphatidylethanolamine vesicles at 40 °C in their L,, liquid crystalline phase at pH 9.5 and in their Hn inverted hexagonal phase at pH 5.0. The quenching of the donor fluorescence by energy transfer is diffusion controlled in all cases, except in the L~ gel phase. The dimensionality and type of constraints imposed on diffusion are different in each case, with the most efficient diffusion-controlled quenching in the hexagonal phase.
Fluorescence decay of DPH in lipid membranes: Influence of the external refractive index
Biophysical Chemistry, 1993
The radiative decay rate of a fluorescent probe in an optically thin layer is known to depend on the orientation of the probe and on the refractive indices inside and outside the layer (W. Lukosz, Phys. Rev. B 22 (1980) 3030). Fluorescent probes in phospholipid bilayer membranes approximate such a system. The natural lifetime is expected to vary with the refractive index of the medium surrounding the bilayer. The lifetime variation with the refractive index depends on the orientation of the fluorescent probe. This can be used to retrieve the second-rank orientational order parameter, (Pz). The fluorescence decay of all-trans 1,6-diphenyl-1,3,5hexatriene in L-cY-dipalmitoyl-phosphatidylchohne large unilamellar vesicles (LUVs) was measured at a temperature well below that of the phase transition. The refractive index of the medium was varied by addition of glycerol or sucrose. The observed change of decay time with the refractive index followed the theoretical prediction. The value of the order parameter, (Pz), recovered is significantly lower than that obtained from fluorescence polarization data. Possible reasons for this disagreement are discussed.
Fluorescence energy transfer studies of transmembrane location` of retinal in purple membrane
Journal of Molecular Biology, 1983
A diffusion-enhanced energy transfer technique was employed for the determination of transmembrane location of the retinal chromophore in the purple membrane. Theoretical considerations showed that the rate of energy transfer from an energy donor embedded within a membrane to acceptors dissolved in solvent could be described by an analytical function of the distance a of closest approach between the donor and acceptor, if the "rapid-diffusion limit" was attained. The criterion for this limit was given by the relation: (Ro) 6 ~ 20DrDa 4, where R o is the characteristic distance of energy transfer, D is the diffusion coefficient of the acceptor and T D is the fluorescence lifetime of the donor in the absence of acceptor. By photo-reduction of the purple membrane with sodium borohydride, the retinal chromophore was converted to a highly fluorescent derivative, which showed a broad emission band in the visible region. From analysis of the fluorescence decay curves of the photo-reduced purple membrane in the presence of various concentrations of cobalt-ethylenediamine tetraacetate (Co-EDTA : energy acceptor), the depth of the chromophore from the membrane surface was estimated to be 8 {_+3) A. This result was supported by investigations of energy transfer processes in a system where the native purple membranes and the photo-reduced membranes were stacked in parallel ; the energy acceptor in this system was the native retinal chromophore.
Journal of Fluorescence, 1993
Measurements of fluorescence depolarization decays are widely used to obtain information about the molecular order and rotational dynamics of fluorescent probe molecules in membrane systems. This information is obtained by least-squares fits of the experimental data to the predictions of physical models for motion. Here we present a critical review of the ways and means of the data analysis and address the question how and why totally different models such as Brownian rotational diffusion and “wobble-in-cone” provide such convincing fits to the fluorescence anistropy decay curves. We show that while these models are useful for investigating the general trends in the behavior of the probe molecules, they fail to describe the underlying motional processes. We propose to remedy this situation with a model in which the probe molecules undergo fast, though restricted local motions within a slowly rotating cage in the lipid bilayer structure. The cage may be envisaged as a free volume cavity between the lipid molecules, so that its position and orientation change with the internal conformational motions of the lipid chains. This approach may be considered to be a synthesis of the wobble-in-cone and Brownian rotational diffusion models. Importantly, this compound motion model appears to provide a consistent picture of fluorescent probe behavior in both oriented lipid bilayers and lipid vesicle systems.
FEBS Letters, 1991
The orientation of the transition dipole moments in the ground state and the M-intermediate of bacteriorhodopsin were determined by time-resolved and steady-state polarized absorption spectroscopy on samples of oriented immobilized purple membranes. The angle between the transition dipole moment and the membrane normal decreases from 66.8+0.5' in the all-rrons ground state to 64. I +O.SO in the 13-h M-state. The light-induced isomerization of the chromophore is thus accompanied by an orientational change of only about 3" out of the plane of the membrane. The absorption anisotropy at 410 nm remains constant over more than 4 decades of time covering both the rise and decay of M. Conformational changes accompanying a sequential M,-+M, transition thus do not affect !he chromophore orientation.
Nanosecond fluorescence anisotropy decays of 1,6-diphenyl-1,3,5-hexatriene in membranes
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1979
Nanosecond decays of the fluorescence anisotropy, r, were studied for the emission of 1,6-diphenyl-l,3,5-hexatriene (DPH) embedded in a series of mixed multilamellar liposomes containing egg yolk phosphatidylcholine, phosphatidylethanolamine and cholesterol in varying molar ratios, as well as in membranes of intact cells and in virus envelopes.
European Biophysics Journal, 2001
The eects of bacteriorhodopsin analogues and the analogues of a bacteriorhodopsin mutant (D96N) on the lateral organization of lipids have been investigated with lipid species with a variety of acyl chain lengths. The analogues, obtained by regeneration of bacterioopsin or mutant opsin with 14-, 12-, 10-, or 8-¯uororetinal, were reconstituted with 1,2-didodecanoyl-sn-glycero-3-phosphocholine, 1,2-ditetradecanoylsn-glycero-3-phosphocholine, 1,2-dihexadecanoyl-snglycero-3-phosphocholine, and 1,2-dioctadecanoyl-snglycero-3-phosphocholine. The phase behavior of the protein-lipid systems was investigated at dierent temperatures and dierent protein/lipid molar ratios by analyzing the¯uorescence and phase properties of the 1acyl-2-[8-(2-anthroyl)octanol]-sn-glycero-3-phosphocholine probe. The (8,10,12)-bacteriorhodopsins had a similar eect on the lipid phase transition to that induced by native bacteriorhodopsin: a rigidifying eect on the three shorter lipid species and a¯uidifying eect on the longest-chain lipids used. The substitution of retinal with 14-¯uororetinal resulted in much stronger eects of the protein on the lipids: a more pronounced up-shift of the lipid phase transition temperature, a rigidifying eect on all the lipids used, and an elongation of the distance over which the hydrophobic thickness of the lipid bilayer was perturbed by the protein. Evidence was provided that retinal contributed to the long-range protein-lipid interactions in bacteriorhodopsin-phosphatidylcholine vesicles. The extent of this contribution was dependent on the retinal structure in close vicinity to the Shi base and on the compactness of the protein structure.