Fluorescence energy transfer in lipid vesicles. A time-resolved analysis using stretched exponentials (original) (raw)
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Biophysical Journal, 2001
The fluorescent probe laurdan has been shown to be sensitive to the vesicle-to-micelle transition of phosphatidylcholine/octylglucoside (M. Paternostre, O. Meyer, C. , Biophys. J. 69:2476 -2488. On the other hand, a study on the photophysics of laurdan in organic solvents has shown that the complex de-excitation pathway of the probe can be described by two successive processes, i.e., an intramolecular charge transfer followed by dielectric relaxation of the solvent if polar. These two excited-state reactions lead to three emitting states, i.e., a locally excited state, a charge transfer state, and a solvent relaxed state (M. Viard, J. Gallay, M. Vincent, B. Robert and M. , Biophys. J. 73:2221-2234. Experiments have been performed using time-resolved fluorescence on the probe inserted in amphiphile aggregates (mixed liposomes, mixed micelles) different in detergent-to-lipid ratios. The results have been compared with those obtained for laurdan inserted in dipalmitoyl phosphatidylcholine liposomes in the gel and in the fluid lamellar phase. Except for laurdan in dipalmitoyl phosphatidylcholine liposomes in the gel lamellar phase, the red part of the emission spectra originates from the de-excitation of the relaxed excited state of laurdan, indicating that indeed the dielectric relaxation process is an important phenomena in the ground-state return pathway of this probe. On the other hand, the maximization entropy method (MEM) analysis of the fluorescence decay recorded in the blue part of the emission spectra indicates that the dielectric relaxation is not the only reaction occurring to the excited state of laurdan. Moreover, the analysis of the fluorescence decays of laurdan inserted in gel lamellar dipalmitoylphosphatidylcholine (DPPC) liposomes indicates excited-state reactions, although dielectric relaxation is impossible. These results are in agreement with the de-excitation pathway determined from laurdan behavior in organic solvent even if, in most of the aggregates studied in this work, the major phenomenon is the dielectric relaxation of the solvent. All along the vesicle-to-micelle transition, we have observed that the lifetime of the relaxed excited state of laurdan continuously decreases probably due to a dynamic quenching process by water molecules. On the other hand, the time constant of the dielectric relaxation process remains almost unchanged in the lamellar part of the transition but abruptly decreases as soon as the first mixed micelle is formed. This decrease is continuous all over the rest of the transition even if it is more pronounced in the mixed liposomes' and mixed micelles' coexistence. The increase of the octylglucoside-to-lipid ratio of the mixed micelles via the change of the size and the shape of the aggregates may facilitate the penetration and the mobility of water molecules. Therefore, during the vesicle-to-micelle transition, laurdan probes the evolution of both the amphiphile packing in the aggregates and the increase of the interface polarity. This study finally shows that the detergent-to-lipid ratio of the mixed micelles is an important parameter to control to limit the penetration and the mobility of water within the amphiphile aggregates and that laurdan is a nice tool to monitor this phenomenon.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1984
Mathematical treatment of this scheme is given yielding an analytical expression for the time dependence of NBD emission intensity. The use of N-NBD-DLPE in the resonance energy transfer measurements offers the advantage of simple chemical synthesis of the fluorescent probe and leads to additional information on transbilayer motion which was not available with the NBD-iabeled iipids used so far.
Biophysical Journal, 1991
The sensitivity of Laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) excitation and emission spectra to the physical state of the membrane arises from dipolar relaxation processes in the membrane region surrounding the Laurdan molecule. Experiments performed using phospholipid vesicles composed of phospholipids with different polar head groups show that this part of the molecule is not responsible for the observed effects. Also, pH titration in the range from pH 4 to 10 shows that the spectral variations are independent of the charge of the polar head. A two-state model of dipolar relaxation is used to qualitatively explain the behavior of Laurdan. It is concluded that the presence of water molecules in the phospholipid matrix are responsible for the spectral properties of Laurdan in the gel phase. In the liquid crystalline phase there is a relaxation process that we attribute to water molecules that can reorientate during the few nanoseconds of the excited state lifetime. The quantitation of lipid phases is obtained using generalized polarization which, after proper choice of excitation and emission wavelengths, satisfies a simple addition rule.
Analytical biochemistry, 1999
A lipid transfer protein, purified from bovine brain (23.7 kDa, 208 amino acids) and specific for glycolipids, has been used to develop a fluorescence resonance energy transfer assay (anthrylvinyl-labeled lipids; energy donors and perylenoyl-labeled lipids; energy acceptors) for monitoring the transfer of lipids between membranes. Small unilamellar vesicles composed of 1 mol% anthrylvinyl-galactosylceramide, 1.5 mol% perylenoyl-triglyceride, and 97.5% 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) served as donor membranes. Acceptor membranes were 100% POPC vesicles. Addition of glycolipid transfer protein to mixtures of donor and acceptor vesicles resulted in increasing emission intensity of anthrylvinyl-galactosylceramide and decreasing emission intensity of the nontransferable perylenoyl-triglyceride as a function of time. The behavior was consistent with anthrylvinyl-galactosylceramide being transferred from donor to acceptor vesicles. The anthrylvinyl and perylenoyl energy transfer pair offers advantages over frequently used energy transfer pairs such as NBD and rhodamine. The anthrylvinyl emission overlaps effectively the perylenoyl excitation spectrum and the fluorescence parameters of the anthrylvinyl fluorophore are nearly independent of the medium polarity. The nonpolar fluorophores are localized in the hydrophobic region of the bilayer thus producing minimal disturbance of the bilayer polar region. Our results indicate that this method is suitable for assay of lipid transfer proteins including mechanistic studies of transfer protein function.
Biochemistry, 1992
We measured the nonradiative fluorescence resonance energy transfer between 7-nitr0-2,1,3benzoxadiazol-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
Fluorescence energy transfer from diphenylhexatriene to bacteriorhodopsin in lipid vesicles
Biophysical Journal, 1983
Fluorescence energy transfer between the donor diphenylhexatriene (DPH) and the acceptor retinal and fluorescence depolarization of DPH are used to test current theories for fluorescence energy transfer in two-dimensional systems and to obtain information on the effect of the intrinsic membrane protein, bacteriorhodopsin, on the order and dynamics of the lipid phase. Increasing the surface concentration of acceptors by raising the protein to lipid ratio leads to a decrease in the mean fluorescence lifetime by up to a factor of four. When the acceptor concentration is reduced at a fixed protein to lipid ratio by photochemical destruction of retinal, the lifetime increases and reaches approximately the value observed in protein-free vesicles when the bleaching is complete. The shape of the decay curve and the dependency of the mean lifetime on the surface concentration of acceptors are in agreement with theoretical predictions for a two-dimensional random distribution of donors and acceptors. From this analysis a distance of closest approach between donors and acceptors of-18 A is obtained, which is close to the effective radius of bacteriorhodopsin (17 A) and consistent with current ideas about the location of retinal in the interior of the protein. In the absence of energy transfer (bleached vesicles), the steady-state fluorescence anisotropy, r, of DPH is considerably lower than in the corresponding unbleached vesicles, indicating that the effect of energy transfer must be taken into account when interpreting r in terms of order and dynamics.
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.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2004
The structural dynamics of the main phase transition of large unilamellar dinervonoylphosphocholine (DNPC) vesicles was investigated by steady state and time-resolved fluorescence spectroscopy of the membrane incorporated fluorescent lipid analog, 1-palmitoyl-2[10-(pyren-1yl)]decanoyl-sn-glycero-3-phosphocholine (PPDPC). These data were supplemented by differential scanning calorimetry (DSC) and fluorescence anisotropy measured for 1-palmitoyl-2-(3-(diphenylhexatrienyl) propanoyl)-sn-glycero-3-phosphocholine (DPHPC). The collected data displayed several discontinuities in the course of the main transition and the pretransition. The discontinuities seen in the fluorescence properties may require modification of the existing models for phospholipid main transition as a first order process. From our previous study on dipalmitoylphosphocholine (DPPC), we concluded the transition to involve a first-order process resulting in the formation of an intermediate phase, which then converts into the liquid crystalline state by a second order process. Changes in the physical properties of the DNPC matrix influencing probe behavior were similar to those reported previously for PPDPC in DPPC. In gel state DNPC [(T À T m ) < À 10] the high values for excimer/monomer emission ratio (I e /I m ) suggest enrichment of the probe in clusters. In this temperature range, excimer fluorescence for PPDPC (mole fraction X PPDPC = 0.02) is described by two formation times up to (T À T m ) c À 10, with a gradual disappearance of the fractional intensity (I R1 ) of the shorter formation time (s R1 ) with increasing temperature up to (T-T m ) c À 10. This would be consistent with the initiation of the bilayer melting at the PPDPC clusters and the subsequent dispersion of the one population of PPDPC domains. A pronounced decrement in I e starts at (T-T m ) = À 10, continuing until T m is reached. No decrease was observed in fluorescence quantum yield in contrast to our previous study on DPPC/PPDPC large unilamellar vesicles (LUVs) [J. Phys. Chem., B 107 (2003) 1251], suggesting that a lack of proper hydrophobic mismatch may prevent the formation of the previously reported PPDPC superlattice. With further increase in temperature and starting at (T À T m ) c À 1, I e , s R2 , and excimer decay times (s D ) reach plateaus while increment in trans ! gauche isomerization continues. This behavior is in keeping with an intermediate phase existing in the temperature range À 1 < (T À T m ) < 4 and transforming into the liquid disordered phase as a second order process, the latter being completed when (T À T m ) ! 4 and corresponding to c 50% of the total transition enthalpy. D change; I e /I m , ratio of pyrene excimer and monomer fluorescence intensity; Int I e , integrated excimer intensity of the time-resolved fluorescence emission; LUV, large unilamellar vesicle; MLV, multilamellar vesicle; PPDPC, 1-palmitoyl-2[10-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine; T, temperature; T m , main phase transition temperature; T p , pretransition temperature; X lipid , mole fraction of the indicated lipid; s R , rise time (excimer formation time); s D , excimer decay time; s M , weighted average monomer lifetime
Biophysical Journal, 1994
a-Sarcin is a fungal cytotoxic protein that inactivates the eukaryotic ribosomes. A kinetic study of the aggregation and lipid mixing promoted by this protein on phosphatdylglycerol (PG) and phosphatdylserine (PS) vesides has been performed. Egg yolk PG, bovine brain PS, dimyristoyi-PG (DMPG) and dimyristoyl-PS (DMPS) vesicles have been considered. The initial rates of the vesicle aggregation induced by the protein have been measured by stopped-flow 900 light scattering. The formation of a vesicle dimer as the initial step of this process was deduced from the second-order dependence of the initial rates on phospholipid concentration. The highest a-sarcin concentration studied did not inhibit the vesicle aggregation, indicating that many protein molecules are involved in the vesicle cross-linking. These are common characteristcs of the initial steps of the aggregation produced by a-sarcin in the four types of phospholipid vesices considered. However, the kinetics of the scattering values revealed that more complex changes occurred in Fe later steps of the aggregation process of egg PG and brain PS vesicles than in those of their synthetic counterparts. a-Sarcin produced lipid mixing in vesiles composed of DMPG or DMPS, which was measured by fluorescence resonance energy transfer assays. A delay in the onset of the process, dependent on the protein concentration, was observed. Measurement of the rates of lipid mixing revealed that the process is first order on phospholipid concentration. Egg PG and brain PS vesices did not show lipd mixing, although they avidly aggregated. However, a-sarcin was able to promote lipid mixing in heterogeneous systems composed of egg PG + DMPG or brain PS + DMPS vesicles. The dilution of the fluorescence probes was faster when these were incorporated into the bilayers made of synthetic phospholipids than were present in those made of natural phospholipids. The bilayer destabilization produced by the protein in the vesices composed of the dimyristoyl-phospholipids should be transmitted to the more stable ones made of natural phospholipids. The obtained results are interpreted in terms of lipid mixing occurring within vesicle aggregates larger than dimer.