Influence of phospholipid species on membrane fluidity: a meta-analysis for a novel phospholipid fluidity index.

Generalized membrane lipid composition determinants of fluidity have been widely investigated, including phospholipid/cholesterol ratio and unsaturation index. Individual phospholipids differ in their physical characteristics, including their interaction with cholesterol and level of unsaturation, emphasizing the importance of examining their individual influence on membrane fluidity. Thus, the purpose of this study was to examine the dominant phospholipids of biological membranes (phosphatidylcholine, PC; phosphatidylethanolamine, PE; sphingomyelin, SM) through a meta-analysis to assess the validity of an inclusive phospholipid fluidity index (PFI = PC/(PE + SM)) as a determinant for membrane fluidity (expressed as polarization of fluorescent probe 1,6 diphenyl-1,3,5-hexatriene) in comparison to previous phospholipid ratios (PC/PE and PC/SM). The results demonstrate that all indices significantly predicted membrane fluidity at 25°C (based on 10-13 data points). In contrast, only PFI approached significance when predicting membrane fluidity at 37°C (P = 0.10 based on five points). As a result, PFI appears to be the only phospholipid index close to significantly predicting membrane fluidity at mammalian physiological temperature. Because this meta-analysis only assessed studies using mammalian membranes, future work should experimentally assess the validity of the PFI utilizing membranes from mammals and a variety of other species and tissues at their respective physiological temperatures.

High-content imaging of neutral lipid droplets with 1,6-diphenylhexatriene.

Neutral lipid droplets (LDs) are dynamic lipid storage organelles found in all eukaryotic cells from yeast to mammals and higher plants. LDs are important to many physiological processes that include basic cellular maintenance, metabolism, and diverse medical pathologies. LD accumulation has been studied extensively by a range of methods, but particularly by microscopy with several fluorescent dyes extensively used for qualitative and quantitative imaging. Here, we compared established LD stains Nile Red and BODIPY 493/503 to the 4', 6-diamidino-2-phenylindole (DAPI)-range dye 1,6-diphenyl-1,3,5-hexatriene (DPH; excitation/emission λmax=350 nm/420 nm) using high-content image analysis. HeLa cells treated with oleic acid or vehicle were used to compare staining patterns between DPH and Nile Red as well as DPH and the LD protein adipophilin. DPH, Nile Red, and BODIPY 493/503 were compared as assay reagents in oleic acid dose-response experiments. Treatment of MCF-7 cells with sodium butyrate was used as a second cellular system for high-content analysis of LD formation. In this experimental context, we demonstrate the compatibility of DPH with GFP, a technical limitation of Nile Red and BODIPY 493/503 dyes. These data show that DPH has comparable sensitivity and specificity to that of Nile Red. Z'-factor analysis of dose-response experiments indicated that DPH and BODIPY 493/503 are well suited for quantitative analysis of LDs for high-throughput screening (HTS) applications.

Cytoplasmic membrane response to copper and nickel in Acidithiobacillus ferrooxidans.

Metal tolerance has been found to vary among Acidithiobacillus ferrooxidans strains and this can impact the efficiency of biomining practices. To explain observed strain variability for differences in metal tolerance we examined the effects of Cu(2+) and Ni(2+) concentrations (1-200 mM) on cytoplasmic membrane properties of two A. ferrooxidans type strains (ATCC 23270 and 19859) and four strains isolated from AMD water around Sudbury, Ontario, Canada. Growth rate, membrane fluidity and phase, determined from the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH), and fatty acid profiles indicated that three different modes of adaptation were present and could separate between strains showing moderate, or high metal tolerance from more sensitive strains. To compensate for the membrane ordering effects of the metals, significant remodelling of the membrane was used to either maintain homeoviscous adaptation in the moderately tolerant strains or to increase membrane fluidity in the sensitive strains. Shifts in the gel-to-liquid crystalline transition temperature in the moderately tolerant strains led to multiple phase transitions, increasing the potential for phase separation and compromised membrane integrity. The metal-tolerant strain however, was able to tolerate increases in membrane order without significant compensation via fatty acid composition. Our multivariate analyses show a common adaptive response which involves changes in the abundant 16:0 and 18:1 fatty acids. However, fatty acid composition and membrane properties showed no difference in response to either copper or nickel suggesting that adaptive response was non-specific and tolerance dependent. We demonstrate that strain variation can be evaluated using differences in membrane properties as intrinsic determinants of metal susceptibility.

Fungicidal effect and the mode of action of piscidin 2 derived from hybrid striped bass.

Piscidin 2 (P2), a 22-residue cationic peptide isolated from the mast cells of hybrid striped bass, has potent antibacterial activities. However, its antifungal properties are not completely understood. In the current study, we investigated the antifungal effects and mode of action of P2. P2 exhibited potent antifungal activity against human pathogenic fungi. To understand the fungicidal properties of P2, we focused on a membrane-active mechanism of the peptide by in vivo and in vitro testing. Flow cytometric analysis using bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC(4)(3)] and protoplast regeneration experiments showed that P2 caused fungal membrane damage. Furthermore, fluorescence analysis using 1,6-diphenyl-1,3,5-hexatriene (DPH) revealed that P2 created pores in fungal membranes. These results were confirmed with dye leakage tests by using liposomes composed of phosphatidylcholine/phosphatidylserine (3:1, w/w), which mimicked fungal membranes. The present study indicated that P2 exerts its fungicidal effects by perturbing membrane activities.

Short-lived fluorescence component of DPH reports on lipid--water interface of biological membranes.

Fluorescence measurements of 1,6-diphenyl-1,3,5-hexatriene (DPH) in large unilamellar phospholipid vesicles were performed to characterize the influence of the membrane physical properties on the short-lived lifetime component of the fluorescence decay. We have found that the short-lived component of DPH significantly shortens when the membrane undergoes a temperature-induced phase transition as it is known for the long-lived component of DPH. We induced membrane phase transitions also by alcohols, which are reported to be distributed different way in the membrane--ethanol close to the membrane-water interface and benzyl alcohol in the membrane core. A different effect of the respective alcohol on the short and long decay component was observed. Both the time-resolved fluorescence spectra of DPH taken during lipid vesicle staining and the lifetime dependences caused by changes of temperature and/or induced by the alcohols show that the short-lived fluorescence originates from the population of dye molecules distributed at the membrane-water interface.

Incorporation of fluorescent probes into PAMAM dendrimers.

Interactions of two fluorescent probes 1-(trimethylammoniumphenyl)-6-phenyl-1,3,5 hexatriene p-toluenesulfonate (TMA-DPH) and 12-(9-anthroyloxy) stearic acid (12-AS) with polyamidoamine (PAMAM) dendrimers were studied. Changes in fluorescence intensity and steady-state fluorescence anisotropy of TMA-DPH and 12-AS were monitored. It was found that 12-AS molecules incorporated into dendrimer cavities whereas TMA-DPH molecules aggregated on the surface of polymer. Dendrimer size had not significant impact on its host properties.

Phosphorylated 1,6-diphenyl-1,3,5-hexatriene.

A lipophilic dye consisting of a (1E,3E,5E)-1,6-diphenyl-1,3,5-hexatriene (DPH) fluorophore attached to a phosphate diester was prepared, and its fluorescence behavior in different solvent systems and in a liposomal membrane bilayer was examined. The key step in the synthesis of the functionalized end of the dye is a Sonogashira coupling of protected iodophenol with propargyl alcohol; the remaining phenyl ring and double bonds of the all-trans polyene core arise from a Wittig reaction with trans-cinnamaldehyde. Like DPH itself, the emission intensity of its phosphorylated derivative is quenched in polar media.

Probing the role of backbone hydrogen bonding in beta-amyloid fibrils with inhibitor peptides containing ester bonds at alternate positions.

Protein-protein interactions are frequently mediated by stable, intermolecular beta-sheets. A number of cytokines and the HIV Protease, for example, dimerize through beta-sheet motifs. Evidence also suggests that the macromolecular assemblies of peptides and proteins in amyloid fibrils are stabilized by intermolecular beta-sheets. In this paper, we report that interfering with the backbone hydrogen bonding of an amyloidgenic peptide (Abeta16-20) by replacing amide bonds with ester bonds prevents the aggregation of the peptide. The ester bonds were incorporated in an alternating fashion so that the peptide presents two unique hydrogen bonding faces when arrayed in an extended, beta-strand conformation; one face of the peptide has normal hydrogen bonding capabilities, but the other face is missing amide protons and its ability to hydrogen bond is severely limited. Analytical ultracentrifugation experiments demonstrate that this ester peptide, Abeta16-20e, is predominantly monomeric under solution conditions, unlike the fibril-forming Abeta16-20 peptide. Abeta16-20e also inhibits the aggregation of the Abeta1-40 peptide and disassembles preformed Abeta1-40 fibrils. These results suggest that backbone hydrogen bonding is critical for the assembly of amyloid fibrils.

Simulated microgravity (SMG) and bacteria.

This past century has been a scientific revolution in the understanding of the cell as the basic unit of life. However an immense paucity of knowledge exists on microbial growth, survival, function and structure in space. However, there are significant constraints placed on conducting biological research in space such as time, available stowage space, trained personnel, power requirements, weight and the possibility of accidental microbiological contamination. One Earth-based approach is to use a modification of a clinostat known as a HARV (high-aspect-ratio-vessel; Synthecon Inc., Houston, Texas, USA) to conduct this research. In this note we describe the use of the HARV to examine the effects of randomized microgravity (RMG) on bacterial growth and membrane polarization.

Probing DNA-cationic lipid interactions with the fluorophore trimethylammonium diphenyl-hexatriene (TMADPH).

The aim of this study is to get a better understanding of DNA-cationic lipid complex formation and its characterization through the properties of the lipid assembly, using fluorescent probes known to have different locations in the vesicle bilayer, 1,6-diphenylhexa-1,3,5-triene (DPH) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMADPH). The location of these two fluorescent probes in the membrane differs; the positive charge of TMADPH is localized close to the water/lipid interface and its fluorophore is present in the upper part of the acyl chain region while DPH (lacking polar group) is embedded deeper in the hydrophobic part of the bilayer. Unilamellar vesicles ( approximately 100 nm size) composed of N-(1-(2, 3-dioleoyloxy)-propyl)-N,N,N-trimethylammonium chloride (DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as a helper lipid (at 1 : 1 mole ratio) were used as a model of cationic liposomes. Both linear and circular DNA gave almost identical results. DNA-/L+ (mole charge ratio of DNA negatively-charged phosphate to positively-charged lipid) ratios have large effects on the measured parameters. The effects monitored through TMADPH are much more striking than those obtained through the use of DPH, suggesting that the major DNA-lipid interaction occurs at the lipid/water interface. The fact that DNA induced much larger changes in TMADPH fluorescence intensity in H2O than in D2O suggests that the changes in the exposure of TMADPH to water and solvent relaxation effects are involved in the interaction. At DNA-/L+>/=1, fluorescence intensity increased concomitantly with a small increase in TMADPH fluorescence anisotropy without much affect in the size of the complex. At DNA-/L+<0.6, fluorescence quenching proportional to DNA-/L+ occurred, as well as a large increase in TMADPH fluorescence anisotropy and in complex size. These results suggest that at low DNA-/L+, negatively-charged DNA condenses positively-charged lipid headgroups, thereby inducing formation of lipid-ordered domains. This phase separation results in membrane defects at the lipid/water interface and increased exposure of the hydrophobic upper parts of the acyl chains to water, as indicated by the quenching of TMADPH. This leads to instability and aggregation/fusion of the DNA-lipid complexes. On the other hand, at DNA-/L+>/=1, the condensing effect is smaller, involving homogeneous lateral condensation of all the lipids, leading to a reduction in water content near the probe, and the DNA-lipid complexes are relatively small and stable.

Location of diphenyl-hexatriene and trimethylammonium-diphenyl-hexatriene in dipalmitoylphosphatidylcholine bilayers by neutron diffraction.

Neutron scattering experiments have been performed on oriented dipalmitoylphosphatidylcholine (DPPC) bilayers containing diphenylhexatriene (DPH) or its trimethylammonium analog (TMA-DPH). DPH and TMA-DPH were either protonated or deuterated in one of the phenyl rings which afforded by using proton-deuterium contrast methods the location of these fluorescent probes in the model membrane. Both probes exhibit bimodal distributions in DPPC. The position, population and orientation in the two sites vary depending upon the physical state of the bilayer (gel or fluid) and the presence or absence of the TMA group. In gel (L beta') phase lipids DPH is located close and parallel to the bilayer surface (site I) and near the bilayer center, oriented at approximately 30 degrees with respect to the normal to the surface (site II). On going to the fluid (L alpha) phase, a distribution of orientations around the parallel to the surface is only observed for site II. Orientation of DPH in site I is unchanged. In the gel phase TMA-DPH is found in a position close and parallel to the bilayer surface (site I) and in a position (site II) oriented at an angle of approximately 25 degrees with respect to the bilayer normal, with the trimethylammonium group anchored in the head group domain. On going to the fluid phase there is a change in molecular orientation of each of the sites. In site I the molecule penetrates deeper in the bilayer and adopts a approximately 20 degrees tilt with respect to the surface, with an orientational distribution of +/- 10 degrees. In site II the molecule becomes perpendicular to the membrane surface. Changes in population of sites, both with DPH and TMA-DPH, are observed on going from low to high temperatures. They are however difficult to quantitate due to experimental conditions. The H2O-2H2O exchange experiments afforded an estimate of the water layer thickness as well as the maximum penetration of water into the interior of the bilayer.

Human red cell membrane fluidity and calcium pump activity in normolipidaemic type II diabetic subjects.

Red cell membrane cholesterol, 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-[(4-trimethylammonium)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) anisotropies and basal and calmodulin-stimulated calcium pump activities were compared in 16 normolipidaemic Type 2 (non-insulin-dependent) diabetic patients and 20 normolipidaemic control subjects using the Mann-Whitney U-test. Serum cholesterol, membrane cholesterol, and membrane DPH and TMA-DPH anisotropies were similar in the two groups but both basal and calmodulin-stimulated calcium pump activities were reduced in the diabetic group: basal activity (median (inter-quartile range), mumol mg-1 h-1) 1.66 (1.18-1.97) vs 2.09 (1.90-2.50), p < 0.005 and calmodulin-stimulated activity 4.19 (3.07-5.48) vs 5.53 (4.70-6.88), p < 0.006. Although there were no correlations between glycaemic control and membrane anisotropy and between glycaemic control and calcium pump activity, the reduction in calcium pump activity is most likely due to a direct effect of diabetes on the calcium pump protein itself.

Influence of cetiedil on erythrocyte membrane microviscosity and acetylcholinesterase activity.

Since one of the cellular targets of cetiedil, a vaso-erythroactive drug, is likely to be the erythrocyte membrane, we have studied the influence of this drug on erythrocyte membrane microviscosity and acetylcholinesterase activity. No effect was evidenced on microviscosity, as measured by fluorescence polarization of light emitted by DPH or TMA-DPH labelling of the lipid bilayer. Cetiedil, however, did lower acetylcholinesterase activity, but it did not directly inhibit this enzyme activity. It can therefore be considered as an amphiphilic drug that perturbs membrane properties without affecting the physical state of the erythrocyte membrane.

Phosphatidic acid affects structural organization of phosphatidylcholine liposomes. A study of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammonium-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) fluorescence decay using distributional analysis.

The fluorescence decay of 1-(4-trimethylammonium-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) was used to study micro-heterogeneity of 1,2-dimyristoyl-3-sn-phosphatidylcholine (DMPC) liposomes and to characterize the effect of phosphatidic acid on the correlation between fluorescence microheterogeneity and membrane permeability. The fluorescence decay, measured using multifrequency phase fluorometry, has been analyzed either by using a model of discrete exponential components or a model of continuous distribution of lifetime values. Both analyses have shown that TMA-DPH decay is characterized by two components: a long one of about 9 ns and a short one of about 5 ns. In the gel phase, at variance with previous DPH studies, the short component was associated with a large fractional intensity. The distributional analysis showed changes of lifetime values and width in correspondence to the calorimetric transitions. The presence of egg phosphatidic acid increased both long lifetime values and distributional width. The use of TMA-DPH as a probe to evaluate membrane heterogeneity using the distributional width is discussed. The effect of phosphatidic acid on the membrane surface and in the hydrophobic core has been related to its structural properties and to its role in water penetration.

Fluorescent detection of lipopolysaccharide interactions with model membranes.

The critical importance of the lipid A moiety of LPS in resistance and pathogenesis in gram negative infections has led to the assumption that LPS interaction with target cells is due to hydrophobic interaction with plasma membranes. However, work from several laboratories, including our own, is consistent with the presence of a cell membrane structure with characteristics of a "receptor". We have proposed a two-step model for LPS-membrane interaction which resolves the two views, and have developed a model system to control the first step (binding to membrane protein) and study the second step (intercalation into lipid bilayer). We examined the interaction of LPS with small unilamellar phosphatidylcholine vesicles labeled in the hydrophobic portion of the bilayer with the fluorescent probe diphenylhexatrine (DPH) and detected changes in the physical properties of the bilayer by measuring DPH fluorescence anisotropy (delta r). We have found that purified, phenol-extracted S. typhimurium LPS interacts with the bilayer as measured by an increase in delta r and conclude that the LPS aggregate coalesced with the lipid bilayer. The greatest change in delta r was achieved with lipid A, Ra-Re glycolipids and diphosphoryl lipid A. Monophosphoryl lipid A and lipid X were less effective. Preparations of wild-type LPS fractionated according to the length of the O-antigen side chain and unfractionated LPS had least effect on delta r. Thus other factors such as serum components or membrane proteins may be necessary to enhance the interaction of LPS with target cells.

Effect of neuraminidase treatment on the lipid fluidity of the intestinal brush-border membranes.

The effect of neuraminidase treatment on the lipid fluidity of the porcine intestinal brush-border membranes was studied using two fluorescence dyes, pyrene and 1,6-diphenyl-1,3,5-hexatriene. By treatment of the membranes with neuraminidase, the fluorescence parameters of pyrene-labeled membranes changed; i.e., a shift of thermal transition temperature, an increase in the fluorescence quenching rate for Tl+ and a decrease in the fluorescence lifetime. These results suggest that the environmental properties around the dye molecules in the membranes change sensitively upon neuraminidase treatment. Perturbation of the lipid domain in the membranes associated with neuraminidase treatment is also demonstrated by a stimulated solubilization of diphenylhexatriene molecules in the membrane lipids, an increased quenching efficiency with Tl+ and a decreased rotational correlation time of diphenylhexatriene-labeled membranes. Based on these results, we conclude that the lipid organization of the membranes is susceptible to neuraminidase treatment and that the membrane lipid fluidity increases by desialylation by the enzyme treatment.

Relationship between ethanol-induced alterations in fluorescence anisotropy and adenylate cyclase activity.

The effects of butanol, ethanol, and ketamine on adenylate cyclase activity and fluorescence anisotropy were determined in membranes prepared from L6 cells. The experiments were designed to test the hypothesis that the effects of ethanol on adenylate cyclase activity are a consequence of ethanol-induced changes in bulk membrane order. Butanol and ethanol elicited concentration-dependent increases in adenylate cyclase activity and caused decreases in the fluorescence anisotropy of diphenylhexatriene. Butanol was more potent than ethanol in reducing fluorescence anisotropy, and it elicited a greater reduction in fluorescence anisotropy than did ethanol. Butanol was also more potent than ethanol in activating adenylate cyclase, but the highest concentration of butanol used caused a smaller increase in enzyme activity than did the highest concentration of ethanol. When the percent change in adenylate cyclase activity was plotted against the percent change in fluorescence anisotropy at each concentration of alcohol, the increase in isoproterenol-stimulated adenylate cyclase activity per unit change in fluorescence polarization was greater with ethanol than with butanol. Ketamine decreased fluorescence anisotropy but, unlike the alcohols, ketamine caused a decrease in adenylate cyclase activity. A reduction in assay temperature attenuated both the ethanol-induced activation of adenylate cyclase activity and the ethanol-induced reduction in fluorescence anisotropy. Although the data are consistent with the theory that ethanol acts upon a hydrophobic region of the membrane to enhance adenylate cyclase activity, activation of the enzyme does not appear to be a consequence of a decrease in bulk membrane order.

Change in the physical state of platelet plasma membranes upon ionophore A23187 activation. A fluorescence polarization study.

Human platelets were isolated and fluorescence-labelled by 1,6-diphenylhexatriene. Diphenylhexatriene was essentially localized in the plasma membrane, as indicated by trinitrobenzenesulfonate-quenching experiments. A decrease of the fluorescence polarization of diphenylhexatriene was observed upon ionophore A23187 addition in the absence of aggregation. 0.3 microM ionophore allowed to reach the maximum rate of the decrease of fluorescence polarization; it also maximally stimulated the light transmission change, the serotonin release and the thromboxane B2 synthesis. The amplitude of the fluorescence polarization decrease was maximum at platelet concentrations between 4 X 10(7) and 7 X 10(7)/ml. The presence of Ca2+ in the medium increased the rate constant of the polarization change. Chlorpromazine (60 microM) completely inhibited this transition, but at 30 microM its inhibitory effect was reversed by Ca2+. The membrane events implied in platelet activation very likely lead to fluidization of the plasma membrane, perhaps by its fusion with the membranes of internal granules which are relatively depleted of cholesterol. Ca2+ plays a central role in the triggering of the observed effects at the membrane level.

A comparative fluorescence polarization study of cis-parinaroyl-phosphatidylcholine and diphenylhexatriene in membranes containing different amounts of cholesterol.

The steady state fluorescence anisotropy (rs) of 1-acyl-2-cis parinaroyl phosphatidylcholine (PnPC) was compared with that of diphenylhexatriene (DPH) in a variety of model- and biological membrane systems. The fluorescence anisotropy of both probes responded similarly to temperature changes and variations in the acyl chain composition in phosphatidylcholine (PC) liposomes. The presence of proteins and cholesterol increased rs for both DPH and PnPC in the biological membranes as compared to the isolated polar membrane lipids. Comparison of DPH and PnPC in dipalmitoyl-PC-liposomes with and without 50 mol% cholesterol, showed at temperatures above the phase transition of pure dipalmitoyl-PC the presence of cholesterol increased the rs-value for DPH strongly, whereas the rs-value for PnPC was much less affected. In the cholesterol-rich erythrocyte membrane as well as in microsomes from Morris hepatoma 7787, which have an increased cholesterol content as compared to normal rat liver microsomes, the rs of DPH was higher than that of PnPC. No large differences between the rs-values of both probes were evident in the normal cholesterol-poor rat liver microsomes. These effects are discussed in terms of structural differences between the probes and variation of cholesterol content. Alterations in the fatty acid composition of PC present in human erythrocyte membranes were introduced with the aid of a PC-specific transfer protein. Fluorescence anisotropy values of both probes hardly changed upon enrichment of the red cell membrane with either dipalmitoyl PC or 1-palmitoyl-2-arachidonyl PC.

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.