Time-dependent Fluorescence Anisotropy Research Articles

Electronic energy transfer in anisotropic systems. Part 5.—Rhodamine–lipid derivatives in model membranes

The electronic energy transfer between molecules located at lipid/water interfaces of a model membrane has been investigated. The donor–donor systems were lipid derivatives of rhodamine B and rhodamine 101 solubilized in vesicles of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). The systems were studied at low and high dye concentrations, as well as at different temperatures by means of steady-state and time-resolved fluorescence spectroscopy. The time-dependent fluorescence anisotropy, r(t), obtained at low dye concentrations is solely determined by rotational motions of the rhodamine moiety and its orientation. The orientation of the rhodamine chromophore with respect to the normal of the membranes was determined by using linear dichroism (LD) spectroscopy. It is found that the electronic absorption and emission transition dipoles of the dyes are preferentially oriented in the plane of the lipid bilayer. At high dye concentration, r(t) depends on rotational motions of the rhodamine dye as well as energy transfer between adjacent chromophores. An analytical model of energy transfer and a semi-empirical approach of r(t) were used to analyse the experimental results. The models and the analysis of experimental data were tested against simulated data. Our results are compatible with energy transfer among chromophores located in the lipid/water interfaces of a lipid bilayer with their transition dipoles oriented parallel to the membranes, and furthermore undergoing rapid reorientation compared with the rate of energy transfer. The model predicts that the rhodamine B group is located in the water region at ca. 3.5 A from the lipid/water boundary, while the rhodamine 101 dyes are located in this boundary. Furthermore, the dye concentrations predicted from the analysis of the experiments agree with those calculated from independent data.

Direct measurements of energy transfer between identical chromophores in solution

The rate of intramolecular electronic excitation energy transfer in 9,9′ bifluorene has been investigated in a variety of solvents, using both time-correlated single photon counting and femtosecond fluorescence upconversion technique. The kinetics of energy transfer were determined in both cases by time dependent fluorescence anisotropy measurements. The energy transfer dynamics between fluorene moieties has been found to occur on a time scale of approximately 600 fs in different solvents and has been correlated with the T2 value calculated from the absorption linewidth and the β value obtained from jet measurements. The dihedral angle between the fluorene moieties was also calculated from the anisotropy measurement and compared with the values obtained from a solution phase NMR determination.

A Comparison of the Molecular Dynamics of Fluorescent Probes in Curved Lipid Vesicles and Planar Multibilayers

Abstract The dynamic behaviour of the probe molecules DPH and TMA-DPH embedded in small unilamellar vesicles and planar multibilayers of POPC has been studied by time-resolved fluorescence depolarization techniques. The molecular dynamics of the probe molecules was analysed in terms of the rotational diffusion model. It is found that analysis of the time-dependent fluorescence anisotropy from the vesicle system yields two distinct, though statistically equivalent solutions. On the other hand the measurements on planar multibilayers can be interpreted unequivocally. It is shown that the order parameters of the probe molecules are higher in the multibilayers than in the vesicles. A reconstruction of the orientational distribution function reveals that the TMA-DPH molecules have a more pronounced tendency to lie with their long axes parallel to the bilayer surface in the curved vesicles than in the planar multibilayers. An intriguing finding is that the reorientational motion of the probes is considerably sl...

A fluorescence decay study of parinaroyl-phosphatidylinositol incorporated into artificial and natural membranes.

Phosphoinositide metabolism in the plasma membrane is linked to transmembrane signal transduction. In this study we have investigated some physical properties (e.g. molecular order and dynamics) of phosphatidylinositol (PI) in various membrane preparations by time-resolved fluorescence techniques, using a synthetic PI derivate with a cis-parinaroyl chain on the sn-2 position. Phospholipid vesicles, normal and denervated rat skeletal muscle sarcolemmal membranes, and acetylcholine receptor rich membranes from Torpedo marmorata were investigated both at 4 degrees C and 20 degrees C. For comparison we have also included 2-parinaroyl-phosphatidylcholine (PC) in this study. The fluorescent lipids were incorporated into the membrane preparations by way of specific phospholipid transfer proteins, to ensure an efficient and non-perturbing insertion of the lipid-probes. In the Torpedo membranes the order parameters measured for the parinaroyl derivatives of both PC and PI were higher than in phospholipid vesicles. For the Torpedo membrane preparations the acyl chain order for the PI was lower than that for PC, whereas the opposite was true for the vesicles. This inversion strongly suggests that PI has different interactions with certain membrane components as compared to PC. This is also suggested by the significantly higher rate of restricted rotation of PI as compared to PC. In contrast to the order parameters, the correlation times were almost identical for both probes and showed little difference between vesicles and the Torpedo membranes. In contrast to Torpedo membranes, the time-dependent fluorescence anisotropy of the two lipid probes in the sarcolemmal membranes showed, after an initial fast decay, a subsequent gradual increase. This phenomenon was satisfactorily analyzed by assuming two populations of probe lipids with distinct lifetimes, rotational correlation times and molecular order. The order parameter of the population with a short lifetime compared with that of phospholipid vesicles, whereas the population with a long lifetime agreed with that of the Torpedo membranes.

A new look at fluorescence depolarization and the dynamics of anisotropic rotational diffusion

Existing derivations of the time-dependent fluorescence anisotropy of an asymmetric molecule constitute a straightforward application of Favro's work on the rotational diffusion equation (RDE), and make no contribution to the elucidation of rotational dynamics as such. A new approach is developed, and the problem formulated in the parlance of conventional reaction kinetics by demonstrating, with the aid of the method of moments, that the RDE is completely equivalent to a set of ordinary linear differential equations, formally identical with those used to describe a first-order series-parallel reaction scheme.

The effect of factor Va on lipid dynamics in mixed phospholipid vesicles as detected by steady-state and time-resolved fluorescence depolarization of diphenylhexatriene.

We have monitored the thermotropic behavior of mixed dimyristoylglycerophosphoserine (Myr2GroPSer)/dimyristoylglycerophosphocholine (Myr2GroPCho) and Myr2GroPSer/dipalmitoylglycerophosphocholine (Pam2GroPCho) vesicles in the presence of blood-clotting factor Va, using 1,6-diphenyl-1,3, 5-hexatriene as a lipid probe. The Ca2+-independent interaction of factor Va with these vesicles caused a small increase (1-2 degrees C) in the phase transition temperature, regardless of whether Myr2GroPChe was the lower or higher-melting component of the mixed vesicles. The major effect of factor Va was to increase the polarization of diphenylhexatriene when the mixed vesicles were in the liquid crystalline phase. The protein did not change the anisotropy in the bilayer gel state. The increase in the polarization value above the transition temperature closely correlated with the amount of phospholipid-bound factor Va, as verified by a direct binding technique. In addition, we found that the affinity of factor Va for Myr2GroPSer/Myr2GroPCho and Myr2GroPSer/Pam2GroPCho greatly increased at temperatures above the transition temperatures. Time-dependent fluorescence anisotropy measurements of diphenylhexatriene embedded in vesicles in the liquid crystalline state give fluorescence decay curves which can best be fitted by two exponential functions with two rotational correlation times and a constant term. Vesicles composed of Myr2GroPSer exhibit more ordering than Myr2GroPCho vesicles. However, the order parameter of mixed vesicles composed of 40% Myr2GroPSer and 60% Myr2GroPCho (mol/mol) approached that of Myr2GroPCho. Factor Va dramatically increased the longer rotational correlation time of diphenylhexatriene embedded in mixed vesicles in the liquid crystalline state from 3.7 ns to about 17 ns. The second rank-order parameter increased only slightly, but the calculated steady-state anisotropy increased by twofold. These results indicate that the acidic phospholipid-dependent binding of factor Va to mixed vesicles has an ordering effect on the acyl chains of the acidic phospholipids in the outer layer, but leaves the bulk of the phospholipids, mainly phosphatidylcholine, unaltered. None of the factor-Va-induced alterations in the anisotropy parameters point to the occurrence of lateral phase separation.

Effect of orientational order on the decay of the fluorescence anisotropy in membrane suspensions. Experimental verification on unilamellar vesicles and lipid/alpha-lactalbumin complexes

Various models for the analysis of time-dependent fluorescence anisotropy measurements were evaluated. The discussion was based on the analysis of pulsed experiments with 1,6-diphenyl-1,3,5-hexatriene embedded in small unilamellar vesicles of dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine and in dimyristoylphosphatidylcholine/alpha-lactalbumin complexes. It was shown that a recently proposed model (Van der Meer, W., H. Pottel, W. Herreman, M. Ameloot, H. Hendrickx, H. Schröder, 1984, Biophys. J., 46:515-523) described the data better than did the earlier suggested cone model (Kinosita K., Jr., S. Kawato, and A. Ikegami, 1977, Biophys. J., 20:289-305). This permitted the use of the new model for the estimation of the second- and fourth-rank order parameters on nonoriented systems. The results indicated that a fraction of the probes was oriented perpendicularly to the preferred direction of the lipids. An increase of the rotational correlation times of the fluorescent probe and a higher order of its environment were detected after the interaction of alpha-lactalbumin with the dimyristoylphosphatidylcholine vesicles at acidic pH at 24.2 degrees C.

Fluidity properties of isolated chloroplast thylakoid lipids

Chloroplast thylakoid lipids have been isolated free of photosynthetic pigments using a combination of high performance liquid and thin layer chromatography. The hydrophobic fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH) has been incorporated into aqueous dispersions of the isolated lipids in order to investigate dynamic and structural properties of the resulting bilayer membranes. Time dependent fluorescence anisotropy decays have been measured and analysed assuming the wobbling-in-cone model (Kinosita et al., Biophys J 20 (1977) 289-305). The DPH fluorescence lifetimes and the static and dynamic fluorescence anisotropy decay parameters for the probe in a total lipid mixture or in pure digalactosyldiacylglycerol (DGDG), changed in a predictable way with increasing temperature (10°-36°C). For a given temperature, it was found that the total lipid mixture was in general less ordered and showed greater dynamic motion as judged from DPH fluorescence anisotropy and compared with the pure DGDG system, although at 36°C differences in dynamic parameters were less evident. Overall the results obtained emphasize the highly fluid nature of thylakoid membrane lipids and give a basis for investigating how intrinsic proteins modify structural and dynamic properties of the in vivo membrane.

Time-dependent fluorescence intensity and depolarization of diphenylhexatriene in micellar complexes of apolipoprotein C-I and dimyristoylglycerophosphocholine.

The lipophilic fluorescent probe diphenylhexatriene was used to probe the lipid order and dynamics in apolipoprotein C-I . dimyristoylglycerophosphocholine (Myr2Gro-P-Cho) complexes. These complexes contain on the average 25 mol Myr2Gro-P-Cho/mol of apolipoprotein C-I, have a molecular weight around 200 000, and appear as discoidal, stacked particles by negative-stain electron microscopy. Steady-state fluorescence polarization of diphenylhexatriene as a function of temperature gives a broadened and shifted phase transition for Myr2Gro-P-Cho from the gel to liquid-crystalline state, with a mid-point around 27 degrees C. Time-dependent fluorescence intensity and anisotropy measurements of the diphenylhexatriene probe at 15 degrees C and 35 degrees C give fluorescence decay curves which can best be fit by two exponential functions, in each case. The fluorescence lifetimes and their fractional amplitudes approach the corresponding parameters in Myr2Gro-P-Cho vesicles and suggest insignificant effects of the protein on the microenvironment and conformations of the probe. The rotational correlation times and their fractional anisotropies indicate similar local motions of the probe in complexes and in vesicles, but reveal a significant ordering effect of the protein at both temperatures. The overall complex rotation at 15 degrees C has a correlation time of 136 +/- 13 ns, consistent with the size (approximately equal to 200 kDa) and shape (disc approximately equal to 5 x 15 nm) of the particle.

Time-dependent decay and anisotropy of fluorescence from diphenylhexatriene embedded in the chloroplast thylakoid membrane

The hydrophobic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene has been incorporated into the membranes of isolated thylakoids, separated granal and stromal lamellae and aqueous dispersions of extracted thylakoid galactolipids. Time-resolved fluorescence decays have been recorded on a nanosecond scale using single-photon counting in order to assess the motional properties of the probe. All the experimental systems used showed biphasic decay kinetics and the anisotropies of the decays have been interpreted in terms of a model for wobbling diffusion confined to a cone. The analysis has given information about dynamic and structural restraints of the lipid acyl chains. In the intact thylakoid membrane the degree of order of the fatty acid acyl chains is higher and their rate of motion slower than for isolated lipids. Even so, the dynamic and structural parameters indicate that the thylakoids can be considered as a relatively fluid membrane system when compared with many other biological membranes, a property which is probably required to facilitate efficient long-range diffusion of lipophilic mobile electron-transport components. It is suggested that the optimization of thylakoid fluidity is linked to regulation of the membrane protein/lipid ratio which is also likely to be responsible for the higher fluidity of stromal membranes relative to those of the grana.

The structure and conformational dynamics of yeast phenylalanine-accepting transfer ribonucleic acid in solution

The solution structure of yeast tRNAPhe was investigated by using ethidium as a fluorescent probe in the D loop and the anticodon loop. For this purpose the dihydrouracils in position 16/17 and wybutine in position 37 were substituted by ethidium. The lifetimes and the time-dependent anisotropy of ethidium fluorescence were measured by pulsed nanosecond fluorometry. The kinetics of the transitions between different states of the tRNAPheEtd derivatives were determined by chemical relaxation measurements. It was found that the ethidium label irrespective of its position exhibits three different states called T1, T2 and T3 characterized by lifetimes tau 1 = 30 ns, tau 2 = 12 ns, and tau 3 = 3 ns. The lifetime differences are due to different accessibilities of ethidium for solvent quenching in the three states. Thus, there are three different defined structural environments of the ethidium in both the anticodon and the D loop. The distribution of the three states was measured as a function of Mg2+ concentration and temperature; it was found that state T3 is favored over states T2 and T1 by both increasing Mg2+ concentration and increasing temperature. The chemical relaxation kinetics exhibit a fast transition between T1 and T2 (10--100 ms) and a slow transition between T2 and T3 (100--1000 ms). The rates of both transitions depend likewise on Mg2+ concentration and temperature. The equilibrium and kinetic data clearly show the presence of strong and weak interactions between Mg2+ and tRNA. A cooperative model accounting for this behavior is developed. The ethidium probe behaves identically when located in different regions of the tRNA regarding both its distribution of states and its transition kinetics. This suggests that the different spectroscopic states report different conformations of the tRNA structure. The dependence of the three states on Mg2+ and spermine indicates that conformation T3 is closely related to or identical with the crystal structure. The rotational diffusion constants indicate that of all three states T3 is most extended while T2 is most compact. The thermodynamic analysis reveals that the strongly bound Mg2+ ions reduce both the activation entropy and enthalpy of all transitions. The weakly bound Mg2+ ions increase both the activation enthalpy and entropy of the slow transition between T2 and T3. It is suggested that the breaking of several intramolecular bonds, e.g., hydrogen bonds, is involved in this transition.