Gel Phase Lipid Research Articles

Aluminum Affects Membrane Physical Properties in Human Neuroblastoma (IMR-32) Cells Both before and after Differentiation

The capacity of Al3+ to induce changes in the physical properties of plasma membrane from human neuroblastoma cells (IMR-32) was investigated, and the magnitude of the changes was compared with that obtained after cell differentiation to a neuronal phenotype. Similarly to our previous results in liposomes, Al3+ (10 to 100 μM) caused a significant loss of membrane fluidity, being the differentiated cells more affected than the nondifferentiated cells. Al3+ also increased the relative content of lipids in gel phase and promoted lipid rearrangement through lateral phase separation, with the magnitude of this effect being similar in nondifferentiated and differentiated cells. Since membrane physical properties depend on bilayer composition, we characterized the content of proteins, phospholipids, cholesterol, and fatty acids in the IMR-32 cells before and after differentiation. Differentiated cells had a significantly higher content of unsaturated fatty acids, creating an environment that favors Al3+-mediated effects on the bilayer fluidity. The neurotoxic effects of Al3+ may be, at least in part, due to alterations of neuronal membrane physical properties, with potential consequences on the normal functioning of membrane-related cellular processes.

Effects of Phospholipid Headgroup and Phase on the Activity of Diacylglycerol Kinase of Escherichia coli

Diacylglycerol kinase (DGK) of Escherichia coli has been reconstituted into a variety of phospholipid bilayers and its activity determined as a function of lipid headgroup structure and phase preference. The anionic phospholipids dioleoylphosphatidic acid, dioleoylphosphatidylserine, and cardiolipin were all found to support activities lower than that supported by dioleoylphosphatidylcholine. In mixtures of dioleoylphosphatidylcholine and 20 mol % anionic phospholipids, the presence of anionic phospholipids all resulted in lower activities than in dioleoylphosphatidylcholine, except for dioleoylphosphatidylglycerol whose presence had little effect on activity. In some cases, the low activity in the presence of anionic phospholipid followed from a decrease in v(max); in some cases, it followed from an increase in the K(m) for diacylglycerol, and in the case of dioleoylphosphatidic acid, it followed from both. Activities in mixtures containing 80 mol % dioleoylphosphatidylethanolamine were lower than in dioleoylphosphatidylcholine at temperatures where both lipids adopted a bilayer phase; at higher temperatures where dioleoylphosphatidylethanolamine preferred a hexagonal H(II) phase, the differences in activity were greater. These experiments suggest that the presence of lipids preferring a hexagonal H(II) phase leads to low activities. Activities of DGK are low in a gel phase lipid.

Equilibrium and Dynamic Interfacial Tension Measurements at Microscopic Interfaces Using a Micropipet Technique. 2. Dynamics of Phospholipid Monolayer Formation and Equilibrium Tensions at the Water−Air Interface

Phospholipid monolayers at the water−air interface have been used extensively as models approximating one-half of the biological bilayer membrane and are of particular interest as stabilizers of microbubbles and emulsions. Interfacial tension is an important measure of adsorption and monolayer formation, but for macroscopic interfaces, equilibrium conditions have been difficult if not impossible to reach, especially over the wide range of temperatures necessary to study homologous series of lipids such as the phosphatidylcholines (C12−C18). By use of a new micropipet technique, however, clean, freshly prepared interfaces on the scale of microns can be rapidly and repeatably produced and exposed to monolayer-forming materials (such as phospholipids and surfactants), the equilibrium condition can be quickly achieved in minutes, and changes in the surface tension by introducing new solutions to the interface (and hence, adsorption dynamics) can be accurately measured and tracked. We have used this technique to study the formation of monolayers of various insoluble surfactant systems (pure and mixed phospholipids including cholesterol and charged lipids) at the water−air interface by measuring equilibrium and dynamic surface tensions. We show that liquid-phase lipid systems spread rapidly (50 mN/m min-1) from vesicle suspensions to form monolayers and reach the same equilibrium surface tension of 25 mN/m. The incorporation of cholesterol to create a liquid ordered phase that dramatically changes the molecular packing, elastic modulus, and tensile strength of bilayers has no effect on the rate or equilibrium surface tension of the monolayers. From the limiting surface tension of 25 mN/m for all liquid lipid monolayers (including cholesterol rich), it appears that the liquid state of the lipid acyl chains is the major influence in determining a monolayer surface tension that is similar to the bulk liquid hydrocarbon/gas surface tension. For single-component lipids in their gel phase, the equilibrium surface tension and rate of monolayer formation decreases as the temperature is lowered below Tm, consistent with a reduced spreading pressure for gel-phase lipids. Finally, unscreened interfaces in deionized water were not readily coated by DOPC/DOPG lipids, while in salt solution, monolayers were readily formed, showing that even in the presence of large amounts of lipid as vesicles in suspension, the water−air interface can remain clean when electrostatically stabilized.

Correlation between the effects of a cationic peptide on the hydration and fluidity of anionic lipid bilayers: a comparative study with sodium ions and cholesterol

The cationic tridecapeptide α-melanocyte stimulating hormone (α-MSH) is known to interact with anionic vesicles of 1,2-dimyristoyl- sn-glycero-3-phosphoglycerol (DMPG), partially penetrating the lipid membrane. In the lipid liquid crystal phase, phospholipid derivatives spin labeled at the different C-atoms along the acyl chain, show that the peptide increases the bilayer packing at all depths. Parallel to that, there is an increase in the probe’s isotropic hyperfine splittings, indicating that the peptide significantly decreases the membrane hydrophobic barrier. Accordingly, it is suggested that the increase in membrane packing yielded by α-MSH is partly due to a greater level of interchain hydration. This result is compared to the increase in packing and decrease in polarity yielded by cholesterol, and the absence of structural or polar alterations with Na +. The latter result shows that the peptide effect is not related to an increase of positive charges at the anionic vesicle surface. Alterations on the lipid bilayer polar profile measured by the nitroxide hyperfine splitting z component in frozen samples are shown to be different from those obtained at room temperature. However, it is shown here that a certain correlation can be drawn between the increase in polarity measured in frozen samples and the packing effect caused by the different molecules in the lipid gel phase.

Effects of Vitrified and Nonvitrified Sugars on Phosphatidylcholine Fluid-to-Gel Phase Transitions

DSC was used to study the ability of glass-forming sugars to affect the gel-to-fluid phase transition temperature, T m, of several phosphatidylcholines during dehydration. In the absence of sugars, T m increased as the lipid dried. Sugars diminished this increase, an effect we explain using the osmotic and volumetric properties of sugars. Sugars vitrifying around fluid phase lipids lowered T m below the transition temperature of the fully hydrated lipid, T o. The extent to which T m was lowered below T o ranged from 12° to 57°, depending on the lipids’ acyl chain composition. Sugars vitrifying around gel phase lipids raised T m during the first heating scan in the calorimeter, then lowered it below T o in subsequent scans of the sample. Ultrasound measurements of the mechanical properties of a typical sugar-glass indicate that it is sufficiently rigid to hinder the lipid gel-to-fluid transition. The effects of vitrification on T m are explained using the two-dimensional Clausius-Clapeyron equation to model the mechanical stress in the lipid bilayer imposed by the glassy matrix. Dextran and polyvinylpyrrolidone (PVP) also vitrified but did not depress T m during drying. Hydration data suggest that the large molecular volumes of these polymers caused their exclusion from the interbilayer space during drying.

Conformation of the Acyl Chains in Diacylphospholipid Gels by IR Spectroscopy

An IR study of the conformational disorder in the part of the hydrocarbon chain nearest the ester group in gel-phase lipid bilayers is presented. The infrared method for determining the conformation of the CH2−CH2 bond attached to the ester group is discussed and applied to a number of representative gel-phase 1,2-diacylphosphatidylcholine fully hydrated dispersions, including symmetric and mixed-chain PCs in their gel and subgel phases. The effect on chain conformation of the headgroups PC, PE, and PA- was explored. Only two conformations of the alkyl chains were found: a planar (all-trans) chain and a nonplanar chain that is all-trans except for the CH2−CH2 bond nearest the ester group. Concentrations of the two conformers were also determined. The overall concentration of the planar form always exceeds 50% but varies significantly depending on the lipid involved. In two cases, the concentration of the planar conformer was estimated for both the SN1 and SN2 chains.

Molecular dynamics simulations of gel (LβI) phase lipid bilayers in constant pressure and constant surface area ensembles

The results of a series of molecular dynamics simulations of the gel state of a dipalmitoylphosphatidylcholine bilayer at 293 K are described. The simulations, ranging from 40 ps to 2.5 ns, show clearly that: a flexible cell geometry is essential during equilibration; Ewald summation of electrostatics is superior to spherical cutoff methods; water exchange with the carbonyl group of chain 2 takes place on the ns time scale, while there is almost no hydration of chain 1. There is overall good agreement (D-spacing, chain tilt, fraction gauche, and area compressibility modulus) with experiment, though the surface area per lipid is slightly underestimated. The randomization of torsion 1 of chain 2 from exclusively gauche minus (as specified in the initial condition modeled from the crystal structure of a related lipid) to a mixture of g+/g− over the course of approximately 2 ns is a critical feature of the study. The torsional equilibration proceeded steadily when simulating at constant surface tension, but was effectively quenched by simulation at constant area. The associated presence of conformational degeneracy of this torsion, and conformational disorder in the upper region of chain 2, is most likely associated with the seemingly anomalous infrared (IR) results for gauche bonds in the upper region of the chains. It may also be a characteristic of the gel phase, and be related to the long time required for the gel to subgel transition.

Nonlinear electron paramagnetic resonance studies of the interaction of cytochrome c oxidase with spin-labeled lipids in gel-phase membranes.

The interaction of lipids, spin-labeled at different positions in the sn-2 chain, with cytochrome c oxidase reconstituted in gel-phase membranes of dimyristoylphosphatidylglycerol has been studied by electron paramagnetic resonance (EPR) spectroscopy. Nonlinear EPR methods, both saturation transfer EPR and progressive saturation EPR, were used. Interaction with the protein largely removes the flexibility gradient of the lipid chains in gel-phase membranes. The rotational mobility of the chain segments is reduced, relative to that for gel-phase lipids, by the intramembranous interaction with cytochrome c oxidase. This holds for all positions of chain labeling, but the relative effect is greater for chain segments closer to the terminal methyl ends. Modification of the paramagnetic metal-ion centers in the protein by binding azide has a pronounced effect on the spin-lattice relaxation of the lipid spin labels. This demonstrates that the centers modified are sufficiently close to the first-shell lipids to give appreciable dipolar interactions and that their vertical location in the membrane is closer to the 5-position than to the 14-position of the lipid chains.

The membrane lipid environment modulates drug interactions with the P-glycoprotein multidrug transporter.

The P-glycoprotein multidrug transporter functions as an ATP-driven efflux pump for a large number of structurally unrelated hydrophobic compounds. Substrates are believed to gain access to the transporter after partitioning into the membrane, rather than from the extracellular aqueous phase. The binding of drug substrates to P-glycoprotein may thus be modulated by the properties of the lipid bilayer. The interactions with P-glycoprotein of two drugs (vinblastine and daunorubicin) and a chemosensitizer (verapamil) were characterized by quenching of purified fluorescently labeled protein in the presence of various phospholipids. Biphasic quench curves were observed for vinblastine and verapamil, suggesting that more than one molecule of these compounds may bind to the transporter simultaneously. All three drugs bound to P-glycoprotein with substantially higher affinity in egg phosphatidylcholine (PC), compared to brain phosphatidylserine (PS) and egg phosphatidylethanolamine (PE). The nature of the lipid acyl chains also modulated binding, with affinity decreasing in the order egg PC > dimyristoyl-PC (DMPC) > dipalmitoyl-PC (DPPC). Following reconstitution of the transporter into DMPC, all three compounds bound to P-glycoprotein with 2-4-fold higher affinity in gel phase lipid relative to liquid-crystalline phase lipid. The P-glycoprotein ATPase stimulation/inhibition profiles for the drugs were also altered in different lipids, in a manner consistent with the observed changes in binding affinity. The ability of the drugs to partition into bilayers of phosphatidylcholines was determined. All of the drugs partitioned much better into egg PC relative to DMPC and DPPC. The binding affinity increased (i.e., the value of Kd decreased) as the drug-lipid partition coefficient increased, supporting the proposal that the effective concentration of the drug substrate in the membrane is important for interaction with the transporter. These results provide support for the vacuum cleaner model of P-glycoprotein action.

An animal virus-derived peptide switches membrane morphology: possible relevance to nodaviral transfection processes.

The N-terminal domain of the capsid protein cleavage product of the flock house virus (FHV) consists of 21 residues and forms an amphipathic alpha-helix, which is thought to play a crucial role in permeabilizing biological membranes for RNA translocation in the host cell. We have found that the Met --> Nle variant of this domain (denoted here as gamma1) efficiently induces the formation of the interdigitated gel phase (LbetaI) of 1, 2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) bilayers. In situ scanning force microscopy of solid supported bilayers and fluorescence spectroscopy of peptide-treated DPPC vesicles provide evidence for the formation of acyl chain interdigitated lipid domains. It could be shown by fluorescence spectroscopy that the peptide inserts in the DPPC matrix above the main transition temperature of the lipid, while the formation of domains with decreased thickness occurs after the sample is cooled to 25 degrees C. The orientation and secondary structure of the peptide in lipid bilayers were investigated using attenuated total reflectance infrared (ATR-IR) and circular dichroism (CD) spectroscopy. These results enabled us to formulate a mechanistic model for the peptide-mediated induction of interdigitation in DPPC bilayers. Moreover, the membrane activity of gamma1 with gel phase lipids established in this study may have further implications for the infection strategy adopted by simple RNA viruses.

Contributions to Maxima in Protein Kinase C Activation

In many lipid systems, the activity of protein kinase C (PKC) exhibits a peak followed by a decline as the mol % of one component is increased. In these systems, an increase in one lipid component is always at the expense of another or accompanied by a change in total lipid concentration. Here we report that in saturated phosphatidylserine (PS)/phosphatidylcholine (PC)/diacylglycerol (DAG) mixtures, increasing PS or DAG at the expense of PC revealed an optimal mol % PS, dependent on mol % DAG, with higher mol % PS diminishing activity. The decrease at high mol % PS is probably not attributable simply to more gel-phase lipid due to the higher melting temperature of saturated PS versus PC because a similar peak in activity occurred in unsaturated lipid systems. Increasing the total lipid concentration at suboptimal mol % PS provided the same activity as higher mol % PS at lower total lipid concentration. However, at optimal mol % PS, activity increased and then decreased as a function of total lipid concentration. PKC autophosphorylation also exhibited an optimum as a function of mol % PS, and increasing the PKC concentration increased the mol % PS at which activity decreased, both for autophosphorylation and for heterologous phosphorylation. Formation of two-dimensional crystals of PKC on lipid monolayers also exhibited a peak as a function of mol % PS, and the unit cell size of the crystals formed shifts from 50 x 50 A at low mol % PS to 75 x 75 A at higher PS. Collectively, these data suggest the existence of optimal lipid compositions for PKC activation, with increased quantity of these domains serving to dilute out enzyme-substrate aggregates and/or enzyme-enzyme aggregates on the lipid surface.

Observation of a Rectangular Columnar Phase in Condensed Lamellar Cationic Lipid-DNA Complexes

We report on a synchrotron x-ray diffraction study of saturated cationic lipid complexed with DNA. In the lipid gel phase L c0 the complexes exhibit Bragg reflections due to lamellar lipid bilayer stacking and three nonintegral peaks in agreement with an intercalated centered rectangular columnar superlattice of DNA. The diffuse broadening of the DNA peaks is caused by in-plane translational disorder of the DNA strands and is approximated by Lorentzians with positional correlation lengths of jy 250 A out of plane and jx . 2000 A in plane. We interpret our results in terms of a system of interacting two-dimensional smectic layers. [S0031-9007(98)07820-X]

Thermal acclimation of phase behavior in plasma membrane lipids of rainbow trout hepatocytes.

The fluorescent probes laurdan (6-dodecanoyl-2-dimethylaminonapthalene) and N-[7-nitrobenz-2-oxa-1, 3-diazol-4-yl] dipalmitoyl-L-alpha-phosphatidylethanolamine (NBD-PE) in addition to Fourier transform infrared spectroscopy (FTIR) were employed to measure the phase behavior and physical properties of hepatocyte plasma membranes isolated from the livers of thermally acclimated (5 and 20 degreesC) rainbow trout (Oncorhynchus mykiss). The primary objective was to determine the extent to which the phase behavior of membrane lipids is conserved at different growth temperatures. Arrhenius plots of laurdan-generalized polarization revealed a single discontinuity believed to reflect either the onset of the gel-fluid phase transition or the formation of gel phase microdomains, and this discontinuity occurred at significantly higher temperatures in membranes of 20 degrees C (13.2 +/- 0.7 degrees C)- than 5 degrees C (7.2 +/- 0.1 degrees C)-acclimated trout. Similarly, acclimation from 5 to 20 degrees C increased both the onset temperature (from 2.0 +/- 0.3 to 7.2 +/- 0.6 degrees C) and the thermal range (from 10.9 +/- 0.5 to 16.0 +/- 1.0) of the gel-fluid transition as assessed by FTIR. The gel-fluid transition midpoint (approximately -2 degrees C) and completion temperatures (-9 degrees C) were unchanged by thermal acclimation. The anisotropy of NBD-PE fluorescence displayed a distinct minimum in membranes of both warm- and cold-acclimated trout (reflecting alterations in lipid packing that in pure lipid membranes ultimately lead to the formation of nonlamellar phases) in the range of 56-58 degrees C; only membranes of 5 degrees C-acclimated trout displayed an additional minimum at significantly lower temperatures (24.5 +/- 1.7 degrees C). Collectively, these data suggest that the regulation of both the temperature at which gel phase lipids begin to form in response to cooling as well as the propensity of membrane lipids to form nonlamellar phases at higher temperatures may be key features of membrane organization subject to adaptive regulation.

Effects of dipalmitoylglycerol and fatty acids on membrane structure and protein kinase C activity

The individual and combined effects of the saturated diacylglycerol (DAG) dipalmitin (DP) and saturated or polyunsaturated unesterified fatty acids (PUFAs) on both the structure of phosphatidylcholine/phosphatidylserine (PC/PS; 4:1 mol/mol) bilayers and on protein kinase C (PKC) activity were studied using 2H nuclear magnetic resonance (NMR) and enzyme activity assays. In the absence of DP, PUFAs only slightly activated PKC whereas palmitic acid had no effect. In the absence of fatty acids, DP induced lateral phase separation of the bilayer into liquid-crystalline and gel phases. Under these conditions virtually all DP was sequestered into the gel phase and no activation of PKC was observed. The addition of polyunsaturated arachidonic or docosahexaenoic acids to the DP-containing bilayers significantly increased the relative amounts of DP and other lipid components in the liquid-crystalline phase, correlating with a dramatic increase in PKC activity. Furthermore, the effect was greater with PS, resulting in an enrichment of PS in the liquid-crystalline domains. In the presence of DP, palmitic acid did not decrease the amount of gel phase lipid and had no effect on PKC activity. The results explain the observed lack of PKC-activating capacity of long-chain saturated DAGs as due to the sequestration of DAG into gel domains wherein it is complexed with phospholipids and thus not available for the required interaction with the enzyme.

Fusion of phospholipid vesicles induced by the ribosome inactivating protein saporin.

The single chain ribosome-inactivating protein Saporin-S6 (SO-6) induces the fusion of acid phospholipid vesicles. The extent of fusion was measured by resonance energy transfer assay between the N-(7-nitro-2-1,3-benzoxadiazol-4-yl)-dimyristoylphosphatidyl lithanolamine (NBD-PE)(donor) and N-(lissamine rhodamine B sulphonyl)-diacylphoshaidylethanolamine (Rh-PE) (acceptor) incorporated in the vesicle. The saturated lipid/protein molar ratio is approx. 100:1. The time course of fusion of vesicles induced by the protein showed that the process was completed within 10 minutes, and the size of the particles in the medium was enlarged which conforms the occurrence of the fusion occurring. The fusion is temperature dependent and the liquid-crystalline state lipid is more apt to fuse than the gel phase lipid. The effect of SO-6 is also dependent on ionic strength and pH, high salt concentration and basic pH may abolish fusion, which suggests that both electrostatic and hydrophobic components may be involved in the process.

Effect of polyunsaturated fatty acid deficiency on dipole relaxation in the membrane interface of rat liver microsomes

The influence of a fat-free diet on the lipid dynamics of rat liver microsomes and liposomes of microsomal lipids was studied by using different fluorescence methods. Lifetime distribution and rotational diffusion of probes with different localization in the lipid bilayer were measured using multifrequency fluorometry. Lateral mobility was studied by measuring excimer formation of pyrenedodecanoic acid. Dipolar relaxation in the interfacial region was studied using 2-dimethylamino-6-lauroylnaphthalene (Laurdan). In spite of large changes in the fatty acid composition of microsomal lipids, polyunsaturated fatty acid deficiency showed no effect on the lifetime distribution and rotational mobility of 1,6-diphenyl-1,3,5-hexatriene (DPH), 1-(4-(trimethylamino)phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), 2-, 7- and 12-(9-anthroiloxy)stearic acids. The treatment did not change the lateral diffusion of pyrenedodecanoic acid, either. However, generalized polarization of Laurdan fluorescence was higher in polyunsaturated fatty acid deficient microsomes as compared to the polyunsaturated fatty acid sufficient ones. This effect was also observed in liposomes of the total microsomal lipids, indicating that the changes in fatty acid composition resulting from polyunsaturated fatty acid deficiency produced a small but significant decrease in the rate of dipolar relaxation in the region of the lipid polar groups of the bilayer. The absence of lipid gel phase domains in rat liver microsomes was also indicated by Laurdan fluorescence features.

Fluid or gel phase lipid bilayers to study peptide-membrane interactions?

Since many studies on peptide-membrane interactions are carried out only with fluid phase lipid bilayers (L α -phase, absence of cholesterol) we have investigated whether this phase is really a suitable model for biological membranes. For this purpose the action of melittin on zwitterionic and negatively charged phospholipid bilayers, in the absence and presence of 30 mol% cholesterol, was investigated by solid state 31P-NMR. From the NMR point of view, it appears that systems composed of a single phospholipid best mimic the sterol-containing system a few degrees below the gel-to-fluid phase transition, i. e., in the rippled phase (P β' ). It is then proposed that a relatively rigid membrane containing local defects, rather than a L α -bilayer, is required as an appropriate model for natural membranes when probing the action of melittin. Such requirements might be crucial when studying peptide-lipid interactions.

2H NMR evidence for dynamic disorder in human skin induced by the penetration enhancer azone

Penetration enhancers mediate trans-dermal drug delivery. The penetration enhancer dodecyl-azacycloheptanone (Azone ®) was characterized in situ with 2H NMR on human stratum corneum treated with either uniformly deuterated Azone ® or penetration enhancer with only the entire chain deuterated, in both cases in propylene glycol. The 2H NMR spectrum at ambient temperature constitutes a single narrow line only 180 Hz wide. This contrasts with the complex lineshapes 1–25 kHz wide commonly observed for methylene deuterons of lipids in a bilayer gel phase. Hence there is no evidence for differences in orientational behaviour between different deuterons at ambient temperature. The narrow line translates into a low overall order parameter S CD < 0.0008, for all deuterons, revealing rapid motion of the entire enhancer with correlation times t c ⪡ 6 × 10 −6 s, in all possible directions. At temperatures of about 150 K a 2H NMR pattern characteristic for a random isotropic powder was observed on samples of stacked stratum corncum sheets at different orientations with respect to the magnetic field. This provides strong evidence for random isotropic freezing of the Azone ® molecules upon cooling. The The 2H NMR data suggest than incubation with Azone ® in propylene glycol provokes dynamic structural disorder of the intercellular lamellar lipid structure throughout the stratum corneum and possibly even the creation of fluid domains involving the intercellular lipids, which may be essential for the penetration enhancing effect.

Thermodynamics of transfer of cholesterol from gel to fluid phases of phospholipid bilayers

The partitioning of cholesterol between gel and fluid phases of model membrane bilayers has been studied by differential scanning calorimetry. The partition coefficients and thermodynamics of transfer between phases of dimyristoyl-, dipalmitoyl-, distearoyl- and diarachidoylphosphatidylcholines were determined using a regular solution model for the partitioning process. The partition coefficient, which is the ratio of cholesterol in fluid to gel phase lipid, varies from 2.3 to 8.4 as the acyl chain length increases from the C 14 to the C 20 bilayer and determined at the phase transition temperature. The enthalpies of transfer increase from −46 to +32 kcal/mol as the chain length increases, and there is a compensating increase in the entropy of transfer. The results are interpreted in terms of a disruption of gel phase lipid by the cholesterol, which for the thinner bilayers is increased because the cholesterol molecule spans across the two leafs of the bilayer. At bilayer thicknesses between 18 and 19 carbons, the cholesterol can fit into one-half of the gel phase bilayer, and the enthalpy becomes positive, suggesting less disruption in the gel phase relative to the thinner bilayers. The data support the interpretation that cholesterol does mediate fluidity by acting to disrupt gel domains of lipid.

Folding intermediates of a beta-barrel membrane protein. Kinetic evidence for a multi-step membrane insertion mechanism.

The mechanism of folding and membrane insertion of integral membrane proteins, including helix bundle and beta-barrel proteins is not well understood. A key question is whether folding and insertion are coupled or separable processes. We have used the beta-barrel outer membrane protein A (OmpA) of Escherichia coli as a model to study the kinetics of folding and insertion into dioleoylphosphatidylcholine (DOPC) bilayers, as a function of temperature by gel electrophoresis, protease digestion, and fluorescence spectroscopy. OmpA was unfolded in 8 M urea solution (without detergent), and refolding and membrane insertion was initiated by rapid dilution of the urea concentration in the presence of phospholipid vesicles. In addition to the kinetically unresolved hydrophobic collapse in water, the time course of refolding of OmpA into DOPC bilayers exhibited three kinetic phases over a large temperature range. The first step was fast (k1 = 0.16 min-1) and not very dependent on temperature. The second step was up to two orders of magnitude slower at low temperatures (2 degrees C), but approached the rate of the first step at higher temperatures (40 degrees C). The activation energy for this process was 46 +/- 4 kJ/mol. A third slow process (k3 = 0.9 x 10(-2) min-1 at 40 degrees C) was observed at the higher temperatures. These results suggest that at least two membrane-bound intermediates exist when OmpA folds and inserts into lipid bilayers. We also show that both membrane-bound intermediates can be stabilized in fluid lipid bilayers at low temperatures. These intermediates share many properties with the adsorbed/partially inserted form of OmpA that was previously characterized in gel phase lipid bilayers [Rodionova et al. (1995) Biochemistry 34, 1921-1929]. Temperature jump experiments demonstrate, that the low-temperature intermediates can be rapidly converted to fully inserted native OmpA. On the basis of these and previous results, we present a simple folding model for beta-barrel membrane proteins, in which folding and membrane insertion are coupled processes which involve at least four kinetically distinguishable steps.