Reorientational dynamics in lipid vesicles and liposomes studied with ESR: effects of hydration, curvature and unsaturation

Electron spin resonance experiments were carried out on 3-doxyl-5 alpha-cholestane spin-label (CSL) molecules embedded in multilamellar liposomes and small unilamellar vesicles (SUVs) of palmitoyloleoylphosphatidylcholine (POPC), dioleoylphosphatidylcholine (DOPC) and dilinoleoylphosphatidylcholine (DLPC). The experimental spectra were analyzed by a numerical solution of the stochastic Liouville equation. Effects of temperature, presence of unsaturated bonds and high bilayer curvature on the dynamic behaviour of the lipid molecules were studied. Our results, combined with results from planar multibilayers with a varying hydration rate (Korstanje et al. (1989) Biochim. Biophys. Acta 980, 225-233), give a consistent picture of the orientational order and rotational dynamics of CSL molecules embedded in lipid matrices with various geometrical configurations. Increase of hydration or temperature reduces molecular ordering and increases molecular dynamics. In highly curved vesicle configurations, SUVs, molecular order is found to be lower than in multilamellar liposomes. In contrast, rotational motion is not affected by increase of curvature. In all lipid configurations studied, increase of the number of unsaturated bonds in the fatty acid chains reduces molecular ordering. We find, however, no effect of unsaturation on the rotational mobility of the CSL probe molecules. These results clearly show that changes in molecular orientational order and reorientational dynamics have to be considered separately, and that they are not necessarily correlated as implied by the common concept of membrane fluidity. Comparing our results with data from a motional narrowing analysis shows that the latter approach seriously overestimates the rate of molecular reorientation.     

Structural and dynamic effects of oxidatively modified phospholipids in unsaturated lipid membranes.

Phospholipid hydroperoxides and phospholipid alcohols are two of the major forms of oxidatively modified phospholipids produced during oxidant stress and lipid peroxidation. The process of lipid peroxidation is known to affect the physiological function of membranes. We, therefore, investigated the effects of lipid peroxidation products on the molecular interactions in membranes. Our study was specifically focused on the effects of lipid peroxidation products on static membrane structure (molecular orientational order) and on the reorientational dynamics of the probe molecules in lipid bilayers. The study was done by performing angle-resolved fluorescence depolarization measurements (AFD) on the fluorescent probe diphenylhexatriene (DPH) and by performing angle-resolved electron spin resonance (A-ESR) measurements on cholestane (CSL) nitroxide spin probes embedded in macroscopically oriented planar bilayers consisting of 2-10% 1-palmitoyl-2-(9/13-hydroperoxylinoleoyl)phosphatidylcholine (PLPC-OOH) or 1-palmitoyl-2-(9/13-hydroxylinoleoyl)phosphatidylcholine (PLPC-OH) in 1-palmitoyl-2-linoleoylphosphatidylcholine (PLPC) or dilinoleoylphosphatidylcholine (DLPC). Both probe molecules have rigid cylindrical geometries and report on the overall molecular order and dynamics. However, being more polar, the nitroxide spin probe CSL is preferentially located near the surface of the membrane, while the less polar fluorescent probe DPH reports preferentially near the central hydrophobic region of the lipid bilayers. The results show that the presence of relatively small amounts of oxidatively modified phospholipids within the PLPC or DLPC membranes causes pronounced structural effects as the molecular orientational order of the probe molecules is strongly decreased. In contrast, the effect on membrane reorientational dynamics is minimal.     

Effects of steroid molecules on the dynamical structure of dioleoylphosphatidylcholine and digalactosyldiacylglycerol bilayers

The ESR spectra of cholestane spin labels (CSL) in dioleoylphosphatidylcholine (DOPC) bilayers containing 20 wt% of cholesterol, 7-dehydrocholesterol, beta-sitosterol, stigmasterol and lanosterol exhibit a marked similarity, thus indicating that these steroids induced the same effects on the lipid bilayer over the temperature range 21-55 degrees C. The incorporation of these steroids into the DOPC bilayers enhances the orientational order of the CSL molecules at every temperature studied, but only induces a pronounced slow-down in their rotational motions at temperatures above 35 degrees C. Similar results were obtained in DOPC/ergosterol multilamellar liposomes, but the changes are now less pronounced than in the other five DOPC/steroid systems. In contrast, the addition of stigmasterol to digalactosyldiacylglycerol (DGDG) bilayers appears to increase the order parameter mean value of P2, without affecting the diffusion coefficients. Furthermore, the incorporation of 7-dehydrocholesterol to DGDG bilayers causes a large enhancement in the orientational order, but has only a small effect on D perpendicular of the CSL molecules. Importantly, this latter effect appears to be independent of temperature. The marked changes in the rates of the rotational motion brought about by the addition of steroids, contrasts with the lack of a significant effect of unsaturation on the bilayer dynamics reported by us previously (Korstanje et al. (1989), Biochim. Biophys. Acta 980, 225-233, and 982, 196-204).     

The membrane fluidity concept revisited by polarized fluorescence spectroscopy on different model membranes containing unsaturated lipids and sterols

Quantitative analysis of time-resolved anisotropy measurements of DPH or TMA-DPH in lipid vesicles yields more than one mathematically correct solution. The solutions differ with respect to the average orientation and to the reorientational dynamics of the probe molecules in the bilayer. This leads to quite opposite results regarding the effects of cholesterol on membrane fluidity. One solution predicts an increase in fluidity, the other a decrease. Angle-resolved fluorescence depolarization (AFD) measurements of probes in oriented lipid bilayers enable determination of the average orientation of the probes in the bilayer and, if the fluorescence decay function is known, of the reorientational dynamics. Analysis of AFD measurements of DPH and TMA-DPH show that increasing unsaturation leads to a decrease in molecular order and a decrease in reorientational dynamics (= fluidity) of the probes. At temperatures above the phase transition of the lipids, the addition of cholesterol causes an increase in molecular order and an increase in reorientational dynamics (= fluidity). The plant sterol stigmaterol, which is structurally closely related to cholesterol, has different effects than cholesterol. The effects vary with the structure of the surrounding lipids. The membrane fluidity concept as it was originally proposed by Chapman attempts to describe the structural and dynamic properties of lipids in a membrane using one single parameter indicated as 'membrane fluidity'. Our results show that it is necessary to distinguish between structural parameters describing molecular order and motion parameters describing molecular dynamics, thus supporting a similar suggestion by Seelig and Seelig. In order to be useful, the membrane fluidity concept has to be limited to the parameters describing molecular dynamics.     

Order and dynamics in the lamellar L alpha and in the hexagonal HII phase. Dioleoylphosphatidylethanolamine studied with angle-resolved fluorescence depolarization.

Fluorescence depolarization techniques are used to determine the molecular order and reorientational dynamics of the probe molecule TMA-DPH embedded in the lamellar L alpha and the hexagonal HII phases of lipid/water mixtures. The thermotropically induced L alpha----HII phase transition of the lipid DOPE is used to obtain macroscopically aligned samples in the hexagonal HII phase at 45 degrees C from samples prepared in the lamellar L alpha phase at 7 degrees C. The interpretation of angle-resolved fluorescence depolarization experiments on these phases, within the framework of the rotational diffusion model, yields the order parameters (P2) and (P4), and the diffusion constants for the reorientational motions. The reorientational motion rates of the TMA-DPH molecules in the hexagonal HII phase are comparable with those in the lamellar L alpha phase. Furthermore, the lateral diffusion of the probe molecule on the surface of the lipid/water cylinder in the hexagonal phase is found to be considerably slower than the reorientational motion.     

Tyrosine and peptide reorientational mobility in polymer solutions: Time-dependent fluorescence anisotropy measurements

The ability of peptides to form biologically active conformations that bind to receptors is governed by their dynamics and their propensity to form stable structures. Such factors are consequently important in the design of peptide drugs. Moreover, the stability of such peptides depends on interactions of the peptide with the surrounding matrix. In this article, we study the effect of the polymer poly(vinyl pyrrolidone) (PVP) on the mobility and orientational dynamics of tyrosine and a model peptide, Val-Tyr-Pro-Asn-Gly-Ala (VYPNGA) in glycerol-water solutions. Orientational dynamics are studied experimentally by time-resolved fluorescence anisotropy decays of tyrosine. The presence of PVP leads to the possibility of a distribution of environments for the peptide. The orientational dynamics of tyrosine show that the probe molecule experiences two very different environments. In one, tyrosine rotational motion is weakly coupled to PVP, while in the other, tyrosine interacts strongly with PVP leading to much slower rotational times. The dynamics of VYPNGA are more complex. Fast intramolecular, localized reorientations of the tyrosine are detected. The temperature dependence of the reorientational dynamics of the tyrosine side chain reveal that these motions are shielded from solvent friction. In contrast, global motions of the peptide are severely restricted by PVP, suggesting the ability of the polymer to restrict peptide mobility.     

The dynamics of protein hydration water: a quantitative comparison of molecular dynamics simulations and neutron-scattering experiments

We present results from an extensive molecular dynamics simulation study of water hydrating the protein Ribonuclease A, at a series of temperatures in cluster, crystal, and powder environments. The dynamics of protein hydration water appear to be very similar in crystal and powder environments at moderate to high hydration levels. Thus, we contend that experiments performed on powder samples are appropriate for discussing hydration water dynamics in native protein environments. Our analysis reveals that simulations performed on cluster models consisting of proteins surrounded by a finite water shell with free boundaries are not appropriate for the study of the solvent dynamics. Detailed comparison to available x-ray diffraction and inelastic neutron-scattering data shows that current generation force fields are capable of accurately reproducing the structural and dynamical observables. On the time scale of tens of picoseconds, at room temperature and high hydration, significant water translational diffusion and rotational motion occur. At low hydration, the water molecules are translationally confined but display appreciable rotational motion. Below the protein dynamical transition temperature, both translational and rotational motions of the water molecules are essentially arrested. Taken together, these results suggest that water translational motion is necessary for the structural relaxation that permits anharmonic and diffusive motions in proteins. Furthermore, it appears that the exchange of protein-water hydrogen bonds by water rotational/librational motion is not sufficient to permit protein structural relaxation. Rather, the complete exchange of protein-bound water molecules by translational displacement seems to be required.     

Slow-motion ESR of cholestane spin labels in planar multibilayers of diacyldigalactosyldiglycerides

The orientation and restricted motion of the cholestane spin label (3-spiro-doxyl-5α-cholestane) incorporated into planar multibilayers of diacyldigalactosyldiglycerides extracted from the thylakoid membranes of chloroplasts from different plant leaves has been studied. The experimental ESR spectra were simulated in terms of the slow-tumbling ESR formalism of Freed and co-workers (Polnaszek, C.F., Bruno, G.V. and Freed, J.H. (1973) J. Chem. Phys. 58, 3185–3199). The analysis shows that the degree of orientational order is low. The spin label molecules undergo a faster reorientational motion about their long molecular axes than perpendicular to them. At room temperature the reorientational rate around the long molecular axis falls within the fast-motional limit, while the reorientation rate of the long axis itself corresponds to the slow-tumbling regime. The results indicate that the motion of the labels in bilayers of diacyldigalactosyldiglycerides is considerably slower than that of the same label incorporated into bilayers of saturated phosphatidylcholines above the main phase transition. Differences between bilayers of diacyldigalactosyldiglycerides extracted from different plant membranes have been observed.     

Orientational order and dynamics of hydration water in a single crystal of bovine pancreatic trypsin inhibitor.

The orientational order and dynamics of the water molecules in form II crystals of bovine pancreatic trypsin inhibitor (BPTI) are studied by (2)H NMR in the temperature range 6-50 degrees C. From the orientation dependence of the single crystal quadrupole splitting and linewidth, the principal components of the motionally averaged quadrupole interaction tensor and the irreducible linewidth components for the orthorhombic crystal are determined. With the aid of water orientations derived from neutron and x-ray diffraction, it is shown that the NMR data can be accounted for by a small number of highly ordered crystal waters, some of which have residence times in the microsecond range. Most of these specific hydration sites must be located at intermolecular contacts. The surface hydration layer that is also present in dilute solution is likely to be only weakly ordered and would then not contribute significantly to the splitting and linewidth from the protein crystal. To probe water dynamics on shorter time scales, the (2)H longitudinal relaxation dispersion is measured for a polycrystalline BPTI sample. The observed dispersion is dominated by rapidly exchanging deuterons in protein side chains, undergoing restricted rotational motions on a time scale of 10 ns.     

Rotational diffusion of a steroid molecule in phosphatidylcholine membranes: effects of alkyl chain length, unsaturation, and cholesterol as studied by a spin-label method

Rotational diffusion of cholestane spin-label (CSL), a sterol analogue, in various phosphatidylcholine (PC)-cholesterol membranes was systematically studied by computer simulation of steady-state ESR spectra as a function of chain length and unsaturation of alkyl chains, cholesterol mole fraction, and temperature for better understanding of phospholipid-cholesterol and cholesterol-cholesterol interactions. CSL motion in the membrane was treated as Brownian rotational diffusion of a rigid rod within the confines of a cone imposed by the membrane environment. The wobbling rotational diffusion constant of the long axis, its activation energy, and the cone angle of the confines are obtained for various membranes in the liquid-crystalline phase. The wobbling diffusion constant decreases in the order dilauroyl-PC greater than dimyristoyl-PC greater than dioleoyl-PC approximately dipalmitoyl-PC greater than distearoyl-PC greater than dioleoyl-PC/cholesterol = 3/1 greater than dioleoyl-PC/cholesterol = 1/1 membranes. Activation energy for the wobbling diffusion of the long axis of CSL is strongly dependent on alkyl chain length, unsaturation, and cholesterol mole fraction. It decreases with decrease in alkyl chain length and by introduction of unsaturation in the alkyl chains. In dioleoylphosphatidylcholine membranes, activation energy decreases by a factor of approximately 3 in the presence of 50 mol % cholesterol. Activation energy for wobbling diffusion of CSL in phosphatidylcholine membranes is smaller than the activation energy for translational diffusion of a phospholipid. The former is more dependent on alkyl chain length and unsaturation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence depolarization study of lamellar liquid crystalline to inverted cylindrical micellar phase transition of phosphatidylethanolamine.

The orientational order and rotational dynamics of 2-[3-(diphenyl-hexatrienyl) propanoyl]-3-palmitoyl-L-alpha- phosphatidylcholine (DPH-PC) embedded in dioleoylphosphatidyl-ethanolamine (DOPE) were studied by fluorescence depolarization technique. Upon increasing the temperature, the calculated wobbling diffusion constant D perpendicular of the fluorescent probe was found to decrease at the lamellar (L alpha) to inverted cylindrical (H II) phase transition (10 degrees C). This suggested that the increased gauche rotamers of the alkene chains in the HII phase imposes a constraint in the wobbling motion of the fluorophore. The calculated ratio of order parameter in the L alpha phase to that in the HII phase was 1.7 and different from the theoretical value of 2.0 as predicted from the change in packing symmetry. This result can be explained by a slightly higher local order parameter of the fluorophore or by the fast rotational diffusion motion of the fluorophore around the symmetry axis of the cylindrical tubes in the HII phase.     

Dynamics and ordering of lipid spin-labels along the coexistence curve of two membrane phases: an ESR study

An analysis of electron spin resonance (ESR) spectra from compositions along the liquid-ordered (L(o)) and liquid-disordered (L(d)) coexistence curve from the brain-sphingomyelin/dioleoylphosphatidylcholine/cholesterol (SPM/DOPC/Chol) model lipid system was performed to characterize the dynamic structure on a molecular level of these coexisting phases. We obtained 200 continuous-wave ESR spectra from glycerophospholipid spin-labels labeled at the 5, 7, 10, 12, 14, and 16 carbon positions of the 2nd acyl chain, a sphingomyelin spin-label labeled at the 14 carbon position of the amide-linked acyl chain, a headgroup-labeled glycerophospholipid, a headgroup-labeled sphingomyelin, and the cholesterol analogue spin-label cholestane all within multi-lamellar vesicle suspensions at room temperature. The spectra were analyzed using the MOMD (microscopic-order macroscopic-disorder) model to provide the rotational diffusion rates and order parameters which characterize the local molecular dynamics in these phases. The analysis also incorporated the known critical point and invariant points of the neighboring three-phase triangle along the coexistence curve. The variation in the molecular dynamic structures of coexisting L(o) and L(d) compositions as one moves toward the critical point is discussed. Based on these results, a molecular model of the L(o) phase is proposed incorporating the "condensing effect" of cholesterol on the phospholipid acyl chain dynamics and ordering and the “umbrella model” of the phospholipid headgroup dynamics and ordering.     

Order and fluidity of lipid membranes as determined by fluorescence anisotropy decay

The fluorescence anisotropy decay of four different probes in bilayers of dimyristoylphosphatidylcholine was measured. The probes are diphenylhexatriene, diphenyloctatetraene, trimethylaminodiphenylhexatriene, and trans-parinaric acid. The data for each probe were analyzed in terms of two orientational order parameters, the ordinary order parameter and a higher one, and two rotational diffusion coefficients. The order parameters are largely independent of probe size, but depend on the position of the probes along the membrane normal, thus reflecting the profile of lipid order. If a probe is located in the plateau region of lipid order, its order parameters are interpreted as representing the rigid-body order of lipids. According to this interpretation, the total lipid order in the plateau region originates about equally from rigid-body order and conformational order. The two order parameters obtained for each probe are used to derive approximate angular distributions of the probe molecules. The diffusion coefficient for rotation about the long molecular axis is found to be infinitely large, indicating unhindered rotation about this axis. The diffusion coefficient for rotation about the short molecular axes is evaluated for a viscosity which results as 0.2 poise. This viscosity for rotational diffusion is an order of magnitude smaller than the viscosity for lateral diffusion indicating that at least two viscosities are required to characterize the fluidity of a lipid membrane.Abbreviations FAD fluorescence anisotropy decay - DMR deuterium magnetic resonance - ESR electron spin resonance - DMPC dimyristoylphosphatidylcholine - DPPC dipalmitoylphosphatidylcholine - DPH 1,6-diphenyl-1,3,5-hexatriene - DPO 1,6-diphenyl-1,3,5,7-octatetraene - TMA-DPH 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene - tPnA trans-parinaric acid - NPN N-phenyl-1-naphthylamine - BBO 2,5-bis(4-biphenylyl)oxazole     

Precision parameters from spin-probe studies of membranes using a partitioning technique. Application to two model membrane vesicles

A new version of the ESR spin probe partitioning method is developed and applied to the study of hydration properties of dimyristoyl-phosphatidylglycerol (DMPG) and dimyristoyl-phosphatidylcholine (DMPC) vesicles as functions of salt concentration and temperature above the lipid phase transition. The small spin probe di-tert-butyl nitroxide (DTBN) is used in order to achieve motionally narrowed Electron Spin Resonance (ESR) spectra which may be analyzed with high precision. The new method relies on the use of the second harmonic display of the ESR spectrum followed by spectral line fitting. Spectral fitting yields precise ESR parameters giving detailed information on the surroundings of the spin probe in both phospholipid and aqueous phases. The nitrogen hyperfine coupling constant of DTBN arising from those probes occupying the vesicles is used to study the hydration of the vesicle surface. The hydration properties of the negatively charged vesicle surface of DMPG vesicles are affected by the addition of salt at all temperatures. In contrast, the hydration of DMPC vesicles does not change with salt concentration at the low temperatures. However, at higher temperatures the hydration properties of DMPC vesicle are affected by salt which is interpreted to be due to the faster motion of the phospholipid molecules. The partitioning of the spin probe increases with salt concentration for both DMPG and DMPC vesicles, while water penetration decreases simultaneously. The spin probe in the phospholipid bilayer exhibits anisotropic motion and the extent of the anisotropy is increased at the higher salt concentrations.     

The fidelity of response by 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene in time-resolved fluorescence anisotropy measurements on lipid vesicles

Time-resolved fluorescence anisotropy measurements on 1⁰-[4-(tri-methylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) molecules in lipid vesicles of palmitoyloleoylphosphatidylcholine (POPC), PC extracted from egg yolk (EggPC), dioleoyl-PC (DOPC), dilinoleoyl-PC (DLPC), phosphatidylglycerol extracted from egg yolk (EggPG), dioleoyl-PG (DOPG), sulfoquinovosyldiacylglycerol (SQDG) and digalactosyl-DG (DGDG) with and without cholesterol are presented. The observed intensity decay curves are analyzed simultaneously in terms of the Brownian rotational diffusion model. The analysis thus yields the isotropic fluorescence decay, the initial anisotropy r (0), the order parameters P ₂ and P ₂ as well as the diffusion coefficient of the long molecular axis. It is shown that increasing unsaturation in the acyl chains of the PC lipids results in an increase in the rotational diffusion rates of the probes and a decrease in the order parameter P ₂. However, the value of P ₂ remains unchanged. The corresponding orientational distribution function of the probes is bimodal, with fractions lying preferentially parallel and perpendicular to the local vesicle surface. Surprisingly, the fraction of probe molecules lying with their long axes parallel to the bilayer surface increases with increasing unsaturation with a concomitant narrowing in the width of the distribution of the fraction lying perpendicular to it. As expected, cholesterol is found to increase the order parameters in all the systems and to suppress the tendency of the molecules to lie parallel to the bilayer surface. Furthermore, the rotational diffusion coefficients of the probes is found to increase in all the systems except for DLPC. Interestingly, the effects of unsaturation on the reorientational dynamics of TMA-DPH molecules in the vesicle systems are opposite to those found in the corresponding planar multibilayers (Deinum et al. 1988), whereas the same cholesterol effect is observed for the two systems. Nevertheless, the TMA-DPH molecules exhibit higher diffusion coefficients in the vesicle than in the planar multibilayer systems. In addition, a unimodal distribution of the probe molecules is found in the multibilayer systems. The differences between the two systems are ascribed to the differences in the radius of curvature and the hydration of the bilayers. Lastly we rationalize the bimodal distribution of the TMA-DPH molecules in the vesicles in terms of their observed partition between the lipid and aqueous phases.Abbreviations DPH 1,6-diphenyl-1,3,5-hexatriene - TMA-DPH 1-[4-(trimethylammonio)-phenyl]-6-phenyl-1,3,5-hexatriene - POPC palmitoyloleoylphosphatidylcholine - EggPC PC extracted from egg yolk - DOPC dioleoyl-PC - DLPC dilinoleoyl-PC - EggPG phosphatidylglycerol extracted from egg yolk - DOPG dioleoyl-PG - SQDG sulfoquinovosyldiacylglycerol - DGDG di-galactosyl-DG - HPTLC high performance thin layer chromatography     

Equilibrium and dynamic structure of large, unilamellar, unsaturated acyl chain phosphatidylcholine vesicles. Higher order analysis of 1,6-diphenyl-1,3,5-hexatriene and 1-[4-(trimethylammonio)phenyl]- 6-phenyl-1,3,5-hexatriene anisotropy decay

Equilibrium and dynamic structural properties of minimally to highly unsaturated acyl chain, large, unilamellar phosphatidylcholine (PC) vesicles have been characterized by the dynamic fluorescence properties of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH). Fluorescence lifetimes and equilibrium and dynamic rotational properties of these probes were analyzed by limited-frequency phase-modulation fluorometry in egg PC, palmitoyloleoyl-PC (POPC), dioleoyl-PC (DOPC), palmitoylarachidonoyl-PC (PAPC), and palmitoyldocosahexaenoyl-PC (P-22:6-PC) vesicles over a temperature range from 5 to 37 degrees C. DPH equilibrium orientational distributions were derived according to a model permitting bimodal orientational distributions in which the parallel probability maximum was aligned parallel to the bilayer normal and the orthogonal probability maximum was oriented parallel to the plane of the bilayer. TMA-DPH orientational distributions were derived according to the same model except that all probability was constrained to the parallel orientation. TMA-DPH fluorescence lifetimes were much more sensitive than those of DPH to variations in acyl chain composition and temperature although the same qualitative behavior was generally observed with both probes. Greater acyl chain unsaturation and higher sample temperatures each gave rise to shorter lifetimes consistent with increased water penetrability into the bilayers. Equilibrium order of the hydrocarbon core (as probed by DPH) and of the interfacial and head group regions of the bilayers (as probed by TMA-DPH) was reduced by increasing levels of unsaturation and by higher sample temperatures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Headgroup hydration and motional order of lipids in lamellar liquid crystalline and inverted hexagonal phases of unsaturated phosphatidylethanolamine--a time-resolved fluorescence study

By the use of frequency domain cross-correlation fluorometry, the fluorescence lifetime of the water soluble probe 8,1-anilinonapthalene sulfonic acid (ANS) in aqueous dispersions of dioleoylphosphatidylethanolamine (DOPE) and phosphatidylethanolamine transphosphatidylated from egg phosphatidylcholine (TPE) was measured. The orientational order parameter and rotational diffusion constant of the lipophilic probe 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) were also determined in TPE dispersions. In agreement with a previous study on DOPE (Cheng (1989) Biophys. J. 55, 1025-1031), abrupt changes in both the order packing and rotational diffusion constant were found at the lamellar liquid crystalline (L alpha) to inverted hexagonal (HII) phase transition of TPE. Owing to the subnanosecond resolution capability of this frequency domain fluorometric technique, the heterogeneous fluorescence decay of ANS was resolved into three distinct components with different decay lifetimes (tau's). They were 0 less than tau less than 0.5 ns, 2 less than tau less than 9 ns and tau greater than 15 ns. These lifetime regions were attributed to the partitioning of ANS into the bulk aqueous medium, the lipid/water interface and the lipid hydrocarbon region, respectively. These classifications of lifetime regions were further supported by the sensitivity of those lifetime components with the solvent isotopic shift of D2O. Similar to the changes of orientational order and rotational diffusion of lipophilic probe, the lifetime and intensity fraction of ANS associated with the lipid/water interfacial region declined abruptly at the L alpha-HII transition of both DOPE and TPE. This observation suggested that a dehydration of the lipid headgroup surface occurs at the L alpha-HII transition. This study provided evidence that both the lipid headgroup surface hydration and the lipid dynamics change drastically as a result of the macroscopic rearrangement of lipids at the L alpha-HII transition.