Emission wavelength-dependent decay of the 9-anthroyloxy-fatty acid membrane probes.

Using the phase-modulation technique, we have measured the fluorescence decay of 2- and 12-(9-anthroyloxy)-stearic acid (2- and 12-AS) and 16-(9-anthroyloxy)-palmitic acid (16-AP) bound to egg phosphatidylcholine vesicles or dissolved in nonpolar solvents. Heterogeneity analysis demonstrates that the decay is generally not monoexponential and exhibits large component variations across it emission spectrum. The mean decay time increases (and in parallel, the steady-state polarization decreases) monotonically with increasing wavelength from values at the blue end. The decay at the red side of the emission spectrum contains an exponential term with a negative amplitude, indicating that emission occurs from intermediates created in the excited-state. This behavior is interpreted as arising from intramolecular fluorophore relaxation occurring on the time scale of the fluorescence lifetime. We believe this to be the first study of wavelength-dependent fluorescent emission which is dominated by an intramolecular relaxation process. Although the three probes exhibit qualitatively similar effects, the emission band variations are greatest for 2-AS and smallest for 16-AP. The differences among the probes are not entirely due to environmental factors as demonstrated, for example, by the emission polarization differences observed in the isotropic solvent paraffin oil. In summary, while these findings point out some of the complexities in the 9-anthroyloxy-fatty acids as membrane probes, they also indicate how these complexities might be used as a sensitive measure of lipid-probe interaction.     

Amphiphilic effects of local anesthetics on rotational mobility in neuronal and model membranes

To provide a basis for studying the molecular mechanism of pharmacological action of local anesthetics, we carried out a study of the membrane actions of tetracaine, bupivacaine, lidocaine, prilocaine and procaine. Fluorescence polarization of 12-(9-anthroyloxy)stearic acid (12-AS) and 2-(9-anthroyloxy)stearic acid (2-AS) were used to examine the effects of local anesthetics on differential rotational mobility between polar region and hydrocarbon interior of synaptosomal plasma membrane vesicles (SPMV) isolated from bovine cerebral cortex, and liposomes of total lipids (SPMVTL) and phospholipids (SPMVPL) extracted from the SPMV. The two membrane components differed with respect to 2 and 12 anthroyloxy stearate (2-AS, 12-AS) probes, indicating that a difference in the membrane fluidity may be present. In a dose-dependent manner, tetracaine, bupivacaine, lidocaine, prilocaine and procaine decreased anisotropy of 12-AS in the hydrocarbon interior of the SPMV, SPMVTL and SPMVPL, but tetracaine, bupivacaine, lidocaine and prilocaine increased anisotropy of 2-AS in the membrane interface. These results indicate that local anesthetics have significant disordering effects on hydrocarbon interior of the SPMV, SPMVTL and SPMVPL, but have significant ordering effects on the membrane interface, and thus they could affect the transport of Na⁺ and K⁺ in nerve membranes, leading to anesthetic action.     

Perturbations induced by alpha- and beta-endosulfan in lipid membranes: a DSC and fluorescence polarization study.

The interaction of alpha- and beta-endosulfan isomers with lipid bilayers was searched by differential scanning calorimetry (DSC) and fluorescence polarization of 2-, 6- and 12-(9-anthroyloxy) stearic acids (2-AS, 6-AS and 12-AS) and 16-(9-anthroyloxy) palmitic acid (16-AP). Both endosulfan isomers, at insecticide/lipid molar ratios ranging from 1/40 to 1/1, shift the phase transition midpoint to lower temperature values and broaden the transition profile of dipalmitoylphosphatidylcholine (DPPC) bilayers. At insecticide/lipid molar ratios of 1/40, the isomers fully abolish the bilayer pretransition. Conversely to beta-endosulfan, alpha-endosulfan promotes a new phase transition, centered at 35.4 degrees C, in addition to the main phase transition of DPPC. Therefore, the alpha-isomer may undergo a heterogeneous distribution in separate domains in the plane of the membrane, whereas the beta-isomer may undergo a homogeneous distribution. Fluorescence polarization data indicate that alpha-endosulfan increases the lipid structural order in the regions probed by 2-AS and decreases it in the regions probed by 6-AS, 12-AS and 16-AP. On the other hand, the beta-isomer produces disordering effects in the upper regions of the bilayers, probed by 2-AS, and ordering in deeper regions, probed by 6-AS, 12-AS and 16-AP, mainly in the gel phase. The incorporation of cholesterol into DPPC bilayers progressively decreases the effects of beta-isomer which are vanished at 20 mol% cholesterol. However, this and higher cholesterol concentrations did not prevent alpha-endosulfan membrane interaction, as revealed by DSC and fluorescence polarization. The distinct effects promoted by alpha- and beta-endosulfan are discussed in terms of molecular orientation and positioning within the bilayer. Apparently, the alpha-isomer preferentially locates closer to the phospholipid headgroups whereas the beta-isomer distributes in deeper domains of the bilayer.     

Localization of cyanine dye binding to brush-border membranes by quenching of n-(9-anthroyloxy) fatty acid probes

The location and orientation of 3,3'-dipropylthiodicarbocyanine (diS-C3-(5)) binding sites in renal brush-border membrane vesicles was examined from the quenching of n-(9-anthroyloxy) fatty acid (n-AS) fluorescence. Based on previous kinetic studies (Cabrini, G. and Verkman, A.S. (1986) J. Membrane Biol. 90, 163-175) monomeric aqueous diS-C3-(5) binds to brush-border membrane vesicles (BBMV) by an initial 6 ms association to form bound monomer, a 30-40 ms equilibrium between bound monomer (M) and bound dimer (D), and a 1-1.3 s translocation of D from the outer to inner membrane leaflet. Based on Stern-Volmer and lifetime analyses, M and D quench the fluorescence of the n-AS probes by a collisional mechanism. At low [diS-C3-(5)]/[BBMV] (R), where M predominates, the n-AS quenching efficiencies (Q) are similar (n = 2-16); at high R, where D predominates, Q increases with n (16 greater than 12 much much greater than 6 greater than 2), suggesting that M is oriented parallel, and D perpendicular, to the phospholipid chains deep within the membrane. Mixture of diS-C3-(5) with brush-border membrane vesicles containing n-AS in a stopped-flow apparatus gave a biexponential fluorescence decrease (excitation 390 nm, emission above 450 nm) with time constants 30-40 ms and 1-1.5 s; there was no 6 ms quenching process. These findings are incorporated into a model in which diS-C3-(5) adheres loosely to the outer membrane surface in 6 ms, binds parallel to the membrane phospholipid in 30-40 ms, dimerizes and rotates by 90 degrees in much less than 30 ms, and translocates to the opposite half of the bilayer in 1-15 s.     

Determination of rotational correlation times from deconvoluted fluorescence anisotropy decay curves. Demonstration with 6,7-dimethyl-8-ribityllumazine and lumazine protein from Photobacterium leiognathi as fluorescent indicators

The experimental and analytical protocols required for obtaining rotational correlation times of biological macromolecules from fluorescence anisotropy decay measurements are described. As an example, the lumazine protein from Photobacterium leiognathi was used. This stable protein (Mr 21 200) contains the noncovalently bound, natural fluorescent marker 6,7-dimethyl-8-ribityllumazine, which has in the bound state a long fluorescence lifetime (tau = 14 ns). Shortening of the fluorescence lifetime to 2.6 ns at room temperature was achieved by addition of the collisional fluorescence quencher potassium iodide. The shortening of tau had virtually no effect on the rotational correlation time of the lumazine protein (phi = 9.4 ns, 19 degrees C). The ability to measure biexponential anisotropy decay was tested by the addition of Photobacterium luciferase (Mr 80 000), which forms an equilibrium complex with lumazine protein. Under the experimental conditions used (2 degrees C) the biexponential anisotropy decay can best be described with correlation times of 20 and 60 ns, representing the uncomplexed and luciferase-associated lumazine proteins, respectively. The unbound 6,7-dimethyl-8-ribityllumazine itself (tau = 9 ns) was used as a model compound for determining correlation times in the picosecond time range. In the latter case rigorous deconvolution from the excitation profile was required to recover the correlation time, which was shorter (100-200 ps) than the measured laser excitation pulse width (500 ps).     

Relative location of anthroyl derivatives of stearic acid in low density lipoproteins

Effective quenching constants (K'sv) of 2-, 7- and 12-(9-anthroyloxy)stearic acid (n-AS) fluorescence in LDL were determined. Spin probes I(m, n) (n = 3, 7, 10, 14) and I- anions were used as quenchers. Quenching of 2-AS and 12-AS fluorescence by I(m, n) was the more effective, the deeper spin probe nitroxyl fragment was located (the greater n was); maximal K'sv value corresponded to I(1,14). By contrast, for 7-AS the quenching by I(12,3) was the most effective. 2-AS and 12-AS spectra maxima and fluorescence polarization were similar. We concluded that the 2-AS chromophore was located deeper in LDL phospholipid monolayer than chromophore of 7-AS (as was the case for 12-AS).     

The effects of cholesterol on the time-resolved emission anisotropy of 12-(9-anthroyloxy)stearic acid in dipalmitoylphosphatidylcholine bilayers

The time-resolved fluorescence emission anisotropy of 12-(9-anthroyloxy)stearic acid (12-AS) and 1,6-diphenyl-1,3,5-hexatriene (DPH) have been measured in dipalmitoylphosphatidylcholine liposomes in the presence and absence of 40 mol% cholesterol at temperatures above and below the phase transition temperature (41°C). By using a synchronously-pumped mode-locked frequency-doubled dye laser and single photon counting detection with an excitation response function of 300 picosecond, rotational correlation times down to less than 1 nanosecond could be resolved. Whereas DPH showed only small changes in the limiting anisotropy on the addition of cholesterol, 12-AS showed significant increases in this parameter with the effect being potentiated at higher temperatures. This difference in behaviour has been attributed to a fluorophore-cholesterol interaction that resulted in a change in the fluorophore geometry. Not only do DPH and 12-AS sense different depolarizing rotations due to the different directions of their emission dipoles but also differ in their lipid interactions which alter their limiting anisotropies. The implication is that the comparison of steady-state anisotropy measurements between chemically identical fluorophores in different lipid environments may be complicated by molecular distortions that change the motions to which the steady-state fluorescence parameters will be sensitive.     

Evaluation of hydralazine and procainamide effects on fibroblast membrane fluidity

In this study the membrane fluidity of fibroblasts under different pharmacological treatment was investigated. Two drugs, hydralazine and procainamide, were used to treat the immortalized mouse NIH 3T3 and hamster B14 fibroblasts. Membrane lipid dynamics was measured by fluorescence spectroscopy and electron spin resonance techniques. Two kinds of fluorescent probes (TMA-DPH and 12-(9-anthroyloxy)-stearic acid (12-AS)) and two spin labels (5-doxylstearic acid (5-DS) and 12-doxylstearic acid (12-DS)) were used to monitor fluidity in the upper polar and in the hydrophobic core regions of the lipid bilayer. The drugs influenced the membrane hydrophobic core, of which hydralazine induced fluidization and procainamide increased the rigidity. The membrane fluidity at the surface of the lipid bilayer was not modified by the drugs which indicates that both drugs intercalated mainly into the inner core of the cell membrane.     

Time-resolved tryptophan fluorescence anisotropy investigation of bacteriophage M13 coat protein in micelles and mixed bilayers

Coat protein of bacteriophage M13 is examined in micelles and vesicles by time-resolved tryptophan fluorescence and anisotropy decay measurements and circular dichroism experiments. Circular dichroism indicates that the coat protein has alpha-helix (60%) and beta-structure (28%) in 700 mM sodium dodecyl sulfate micelles and predominantly beta-structure (94%) in mixed dimyristoylphosphatidylcholine/dimyristoylphosphatidic acid (80/20 w/w) small unilamellar vesicles. The fluorescence decay at 344 nm of the single tryptophan in the coat protein after excitation at 295 or 300 nm is a triple exponential. In the micelles the anisotropy decay is a double exponential. A short, temperature-independent correlation time of 0.5 +/- 0.2 ns reflects a rapid depolarization process within the coat protein. The overall rotation of the coat protein-detergent complex is observed in the decay as a longer correlation time of 9.8 +/- 0.5 ns (at 20 degrees C) and has a temperature dependence that satisfies the Stokes-Einstein relation. In vesicles at all lipid to protein molar ratios in the range from 20 to 410, the calculated order parameter is constant with a value of 0.7 +/- 0.1 from 10 to 40 degrees C, although the lipids undergo the gel to liquid-crystalline phase transition. The longer correlation time decreases gradually on increasing temperature. This effect probably arises from an increasing segmental mobility within the coat protein. The results are consistent with a model in which the coat protein has a beta-structure and the tryptophan indole rings do not experience the motion of the lipids in the bilayer because of protein-protein aggregation.     

Time-resolved fluorescence study of the single tryptophans of engineered skeletal muscle troponin C.

The regulatory domain of troponin C (TnC) from chicken skeletal muscle was studied using genetically generated mutants which contained a single tryptophan at positions 22, 52, and 90. The quantum yields of Trp-22 are 0.33 and 0.25 in the presence of Mg2+ (2-Mg state) and Ca2+ (4-Ca state), respectively. The large quantum yield of the 2-Mg state is due to a relatively small nonradiative decay rate and consistent with the emission peak at 331 nm. The intensity decay of this state is monoexponential with a single lifetime of 5.65 ns, independent of wavelength. In the 4-Ca state, the decay is biexponential with the mean of the two lifetimes increasing from 4.54 to 4.92 ns across the emission band. The decay-associated spectrum of the short lifetime is red-shifted by 19 nm relative to the steady-state spectrum. The decay of Trp-52 is biexponential in the 2-Mg state and triexponential in the 4-Ca state. The decay of Trp-90 requires three exponential terms for a satisfactory fit, but can be fitted with two exponential terms in the 4-Ca state. The lower quantum yields (< 0.15) of these two tryptophans are due to a combination of smaller radiative and larger nonradiative decay rates. The results from Trp-22 suggest a homogeneous ground-state indole ring in the absence of bound Ca2+ at the regulatory sites and a ground-state heterogeneity induced by activator Ca2+. The Ca(2+)-induced environmental changes of Trp-52 and Trp-90 deviate from those predicted by a modeled structure of the 4-Ca state. The anisotropy decays of all three tryptophans show two rotational correlation times. The long correlation times (phi 1 = 8.1-8.3 ns) derived from Trp-22 and Trp-90 suggest an asymmetric hydrodynamic shape. TnC becomes more asymmetric upon binding activator Ca2+ (phi 1 = 10.1-11.6 ns). The values of phi 1 obtained from Trp-52 are 3-4 ns shorter than those from Trp-22 and Trp-90, and these reduced correlation times may be related to the mobility of the residue and/or local segmental flexibility.     

Transverse location of anthracyclines in lipid bilayers. Paramagnetic quenching studies

Quenching of anthracycline fluorescence by a series of spin-labeled fatty acids was used to probe the transverse location of the drug in phosphatidylcholine bilayers in the form of small unilamellar vesicles. Stern-Volmer plots of the quenching data indicate that the fluorophore moiety of the anthracycline is intercalated into the hydrocarbon region of the bilayer, with deeper penetration observed in fluid-phase than in solid-phase vesicles. 31P-NMR parameters (T1 and nuclear Overhauser enhancement (NOE] are unaffected by the presence of drug, consistent with a binding site removed from the interfacial region. Comparison of intensity (F0/F) plots with lifetime (tau 0/tau) data shows that the predominant mechanism of anthracycline quenching by membrane-bound nitroxides is static. Since the membrane-bound drug is also accessible to quenching by I-, the binding site in the membrane must create a channel which is accessible to solvent. Two other fluorescent probes, 12-(9-anthroyloxy)stearate (12-AS) and diphenylhexatriene (DPH), were employed to confirm the results obtained with the anthracyclines, giving quenching data representative of their location in the bilayer.     

Picosecond-resolved fluorescence spectra of D-amino-acid oxidase. A new fluorescent species of the coenzyme

Protein dynamics of D-amino-acid oxidase in the picosecond region was investigated by measuring time-resolved fluorescence of the bound coenzyme, FAD. The observed nonexponential fluorescence decay curves were analyzed with four-exponential decay functions. The fluorescence lifetimes at the best fit were 26.6 +/- 0.7 ps, 44.0 +/- 4.2 ps, 177 +/- 11 ps, and 2.28 +/- 0.21 ns at 20 degrees C and 25.2 +/- 3.0 ps, 50.3 +/- 8.7 ps, 228 +/- 27 ps, and 2.75 +/- 0.33 ns at 5 degrees C. Component fractions with the shortest lifetime, ca. 26 ps, were always negative and close to -1. The other fluorescent components of the lifetimes, ca. 47 ps, 200 ps, and 2.6 ns, with positive fractions were assigned to different forms of the enzyme including the dimer, the monomer, and free FAD dissociated from the enzyme. Measurements of the time-resolved fluorescence spectra revealed that the maximum wavelengths of the spectra shifted toward shorter wavelength by 65 nm at 20 degrees C and 36 nm at 5 degrees C within 100 ps after pulsed excitation. The remarkable blue shift was not observed in free FAD. The first spectra immediately after the excitation of the enzyme exhibited maximum wavelengths of 584 nm at 20 degrees C and 557 nm at 5 degrees C. The fluorescence spectra obtained at times later than 100 ps are in good agreement with the one obtained under steady-state excitation of D-amino-acid oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Self-association of the polyene antibiotic nystatin in dipalmitoylphosphatidylcholine vesicles: a time-resolved fluorescence study.

The interaction between Nystatin and small unilamellar vesicles of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, both in gel (T = 21 degrees C) and in liquid-crystalline (T = 45 degrees C) phases, was studied by steady-state and time-resolved fluorescence measurements by taking advantage of the intrinsic tetraene fluorophore present in this antibiotic. It was shown that Nystatin aggregates in aqueous solution with a critical concentration of 3 microM. The enhancement in the fluorescence intensity of the antibiotic was applied to study the membrane binding of Nystatin, and it was shown that the antibiotic had an almost fivefold higher partition coefficient for the vesicles in a gel (P = (1.4 +/- 0.1) x 10(3)) than in a liquid-crystalline phase (P = (2.9 +/- 0.1) x 10(2)). Moreover, a time-resolved fluorescence study was used to examine Nystatin aggregation in the membrane. The emission decay kinetics of Nystatin was described by three and two exponentials in the lipid membrane at 21 degrees C and 45 degrees C, respectively. Nystatin mean fluorescence lifetime is concentration-dependent in gel phase lipids, increasing steeply from 11 to 33 ns at an antibiotic concentration of 5-6 microM, but the fluorescence decay parameters of Nystatin were unvarying with the antibiotic concentration in fluid lipids. These results provide evidence for the formation of strongly fluorescent antibiotic aggregates in gel-phase membrane, an interpretation that is at variance with a previous study. However, no antibiotic self-association was detected in a liquid-crystalline lipid bilayer within the antibiotic concentration range studied (0-14 microM).     

Lipid-phase structure in epithelial cell membranes: comparison of renal brush border and basolateral membranes

The lipid-phase structures of brush border membrane vesicles (BBMV) and basolateral membrane vesicles (BLMV) isolated from rabbit renal cortex were compared by steady-state and phase-modulation measurements of diphenylhexatriene (DPH) and trans- and cis-parinaric acid (tPnA and cPnA) fluorescence. A temperature-scanning system was used which gave reproducible temperature profiles of steady-state and dynamic fluorescence parameters with a resolution of 0.1 degrees C. Steady-state anisotropy of DPH showed a triphasic dependence on temperature with slope discontinuities at 22 +/- 4 and 47 +/- 3 degrees C (BBMV) and at 23 +/- 2 and 48 +/- 1 degrees C (BLMV). At all temperatures, DPH anisotropy in BBMV was greater than that in BLMV. Ground-state heterogeneity analysis of tPnA and cPnA fluorescence lifetime data demonstrated the presence of long (greater than 12 ns) and short (less than 5 ns) lifetime components, interpreted in terms of solid-phase and fluid-phase lipid domains. The fraction of solid-phase phospholipid decreased from 0.9 to 0.1 for BBMV and from 0.7 to 0.3 in BLMV with increasing temperature (10-50 degrees C). In both membranes, tryptophan-PnA fluorescence energy-transfer measurements showed that membrane proteins were surrounded by a fluidlike phospholipid phase. These results demonstrate the inadequacy of steady-state DPH anisotropy data in defining the structural characteristics of complex biological membranes. Results obtained with the phase-sensitive parinaric acid probes demonstrate major differences in the phase structure of the two opposing cell membranes in both the bulk lipid and the lipid microenvironment around membrane proteins.     

Interaction of fluorescence quenchers with the n-(9-anthroyloxy) fatty acid membrane probes

The fluorescence quenching of the $\text{n-(9-}\text{anthroyloxy}\text{)}$ (AO) fatty acid probes has been investigated in aqueous dispersions, vesicles of egg phosphatidylcholine and vesicles formed from red cell ghosts. Negatively charged (KI), neutral (acrylamide) and positively charged (CuSO₄) quenchers were used to monitor the location of the probes. The fluorescence of the probes, with the exception of the shortest chain (11-(9-anthroyloxy)undecanoic acid) is not quenched by acrylamide when associated with vesicles. This indicates that in association with vesicles, the 9-anthroyloxy moiety of the long chain probes is buried within the hydrocarbon region and thus well shielded from the aqueous phase. Measurements with KI indicate that the probes are present in the membrane at depths corresponding to the position of the 9-anthroyloxy moiety on the fatty acid, and that the quencher itself forms a concentration gradient within the membrane. Very little or no CuSO₄ quenching was observed for $\text{n-}\text{(9-anthroyloxy)stearic}$ acid probes $\text{(n-}\text{AS)with}\text{n > 2}$, suggesting that in these vesicles Cu²⁺ does not significantly penetrate the bilayer.     

Structure and dynamics of motilin. Time-resolved fluorescence of peptide hormone with single tyrosine residue.

Time-resolved fluorescence and CD spectroscopy were used to characterize the structure and dynamics of the peptide hormone motilin with a single tyrosine residue among its 22 amino acids. CD spectroscopy showed that secondary structure is independent of concentration in the range 1 x 10(-5)-2.6 x 10(-4) M, and of the presence of DOPC lipid vesicles, but is strongly induced by addition of hexafluoroisopropanol. The fluorescence studies with tyrosine as the intrinsic fluorophore, performed at the MAX synchrotron laboratory at Lund, showed that three fluorescence lifetimes (0.4 ns, 1.7 ns and 3.6 ns at 20 degrees C) and two rotational correlation times (0.4 ns and 5 ns at 20 degrees C) were needed to account for the data. The different decay times are interpreted as representing ground-state rotamers interconverting slowly on the ns time scale. The rotational correlation times are ascribed to local angular motion of the tyrosyl ring, and global motion of the whole peptide, respectively.     

Kinetic model of protein-mediated ligand transport: influence of soluble binding proteins on the intermembrane diffusion of a fluorescent fatty acid

The mechanism (or mechanisms) whereby fatty acids and other amphipathic compounds are transported from the plasma membrane to intracellular sites of biotransformation remains poorly defined. In an attempt to better characterize the role of cytosolic binding proteins in this process, a kinetic model of intermembrane ligand transport was developed in which diffusional transfer of ligand between membrane and protein is assumed. The model was tested by utilizing stopped-flow techniques to monitor the transfer of the fluorescent fatty acid analogue, 12-anthroyloxy stearate (12-AS), between model membrane vesicles. Studies were conducted in the presence or absence of bovine serum albumin (BSA), liver fatty acid-binding protein (L-FABP), and intestinal fatty acid-binding protein (I-FABP) in order to determine the effect of soluble proteins on the rate of intermembrane ligand transfer. As predicted by the model, the initial velocity of 12-AS arrival at the acceptor membrane increases in an asymptotic manner with the acceptor concentration. Furthermore, probe transfer velocity was found to decline asymptotically with increasing concentrations of BSA or L-FABP, proteins that exhibit diffusional transfer kinetics. This observation was found to hold true independent of whether donor or acceptor vesicles were preequilibrated with the protein. In contrast, 12-AS transfer velocity exhibited a linear correlation with the concentration of I-FABP, a protein that is thought to transport fatty acids, at least in part, via a collisional mechanism. Taken together, these findings validate the derived kinetic model of protein-mediated ligand transport and further suggest that the mechanism of ligand-protein interaction is a key determinant of the effect of cytosolic proteins on intracellular ligand diffusion.     

Effect of proteins on fluorophore lifetime heterogeneity in lipid bilayers.

The effect of three different membrane proteins on the fluorescence lifetime heterogeneity of 1,6-diphenyl-1,3,5-hexatriene (DPH) in phospholipid vesicle systems was investigated. For large unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) at 37 degrees C, the fluorescence decay was essentially monoexponential (8.6 and 8.2 ns, respectively) except for a minor component typical of DPH. For gramicidin D reconstituted into DMPC vesicles at a protein/lipid molar ratio of 1/7, the most appropriate analysis of the data was found to be in the form of a bimodal Lorentzian distribution. Centers of the major lifetime components were almost identical with those recovered for vesicles without proteins, while broad distributional widths of some 4.0 ns were recovered. Variation of the protein/lipid molar ratio in sonicated POPC vesicles revealed an abrupt increase in distributional width at ratios approximating 1/15-1/20, which leveled off at about 2.5 ns. For bacteriorhodopsin in DMPC vesicles and cytochrome b5 in POPC, the most appropriate analysis of the data was again found to be in the form of a bimodal Lorentzian also with broad distributional widths in the major component. Lifetime centers were decreased for these proteins due to fluorescence energy transfer to the retinal of the bacteriorhodopsin and heme of the cytochrome b5. Fluorescence energy transfer is distance dependent, and since a range of donor-acceptor distances would be expected in a membrane, lifetime distributions should therefore be recovered independently of other effects for proteins possessing acceptor chromophores.(ABSTRACT TRUNCATED AT 250 WORDS)     

pH-induced reorganization of synaptic membrane as revealed by fluorescence anisotropy and energy transfer

Two fluorescent probes, 2-(9-anthroyloxy)stearate and 12-(9-anthroyloxy)stearate, were used to investigate the effects of the neutralization of membrane charges on the organization of synaptic plasma membrane. Steady state fluorescence anisotropy measurements showed that a pH decrease provoked a rigidification of the synaptic membrane surface, whereas the bilayer core remained unaffected. The same effect was observed with negatively charged lipid vesicles. The relative distribution of proteins and the probes was estimated by fluorescence energy transfer from protein tryptophans to fluorescent probes: a pH decrease provoked an increase of the energy transfer, which was most pronounced with the surface probe, indicating an average closer packing between proteins and the probes. The modifications induced by a pH decrease were temperature dependent and were most marked at low temperatures. The results suggest that neutralization of the membrane charges provoked a redistribution of both membrane lipids and proteins. These findings are discussed in terms of a heterogeneous distribution of these membrane components.     

Application of a reference convolution method to tryptophan fluorescence in proteins. A refined description of rotational dynamics

A reference method for the deconvolution of polarized fluorescence decay data is described. Fluorescence lifetime determinations for p-terphenyl, p-bis[2-(5-phenyloxazolyl)]benzene and N-acetyltryptophanamide (AcTrpNH2) show that with this method more reliable fits of the decays can be made than with the scatterer method, which is most frequently used. Analysis of the AcTrpNH2 decay with p-terphenyl as the reference compound yields an excellent fit with lifetimes of 2.985 ns for AcTrpNH2 and 1.099 ns for p-terphenyl (20 degrees C), whereas the AcTrpNH2 decay cannot be satisfactorily fitted when the scatterer method is used. The frequency of the detected photons is varied to determine the conditions where pulse pile-up starts to affect the measured decays. At detection frequencies of 5 kHz and 15 kHz, which corresponds to 1.7% and 5% respectively of the rate of the excitation photons no effects are found. Decays measured at 30 kHz (10%) are distorted, indicating that pile-up effects play a role at this frequency. The fluorescence and fluorescence anisotropy decays of the tryptophan residues in the proteins human serum albumin, horse liver alcohol dehydrogenase and lysozyme have been reanalysed with the reference method. The single tryptophan residue of the albumin is shown to be characterized by a triple-exponential fluorescence decay. The anisotropy decay of albumin was found to be mono-exponential with a rotational correlation time of 26 ns (20 degrees C). The alcohol dehydrogenase has two different tryptophan residues to which single lifetimes are assigned. It is found that the rotational correlation time for the dehydrogenase changes with excitation wavelength (33 ns for lambda ex = 295 nm and 36 ns for lambda ex = 300 nm at 20 degrees C), indicating a nonspherical protein molecule. Lysozyme has six tryptophan residues, which give rise to a triple-exponential fluorescence decay. A single-exponential decay with a rotational correlation time of 3.8 ns is found for the anisotropy. This correlation time is significantly shorter than that arising from the overall rotation and probably originates from intramolecular, segmental motion.