Effect of the HIV-1 fusion peptide on the mechanical properties and leaflet coupling of lipid bilayers.

The fusion peptide (FP) of the human immunodeficiency virus (HIV) is part of the N-terminus of the viral envelope glycoprotein gp41 and is believed to play an important role in the viral entry process. To understand the immediate effect of this peptide on the cell membrane, we have studied the influence of the synthetic FP sequence FP23 on the mechanical properties of model lipid bilayers. For this purpose, giant unilamellar vesicles were prepared from the unsaturated lipid dioleoylphosphatidylcholine mixed in various molar ratios with FP23. The bending stiffness of the vesicles was measured with two different methods: fluctuation analysis and aspiration with micropipettes. The data obtained from both of these approaches show that the bending stiffness of the membrane decreases gradually with increasing concentration of the FP23 in the bilayer. Low concentrations of only a few mol% FP23 are sufficient to decrease the bending stiffness of the lipid bilayer by about a factor of 2. Finally, data obtained for the stretching elasticity modulus of the membrane suggest that the peptide insertion decreases the coupling between the two leaflets of the bilayer.

The thermotropic phase behavior of cationic lipids: calorimetric, infrared spectroscopic and X-ray diffraction studies of lipid bilayer membranes composed of 1,2-di-O-myristoyl-3-N,N,N-trimethylaminopropane (DM-TAP).

The thermotropic phase behavior of lipid bilayer model membranes composed of the cationic lipid 1,2-di-O-myristoyl-3-N,N,N-trimethylaminopropane (DM-TAP) was examined by differential scanning calorimetry, infrared spectroscopy and X-ray diffraction. Aqueous dispersions of this lipid exhibit a highly energetic endothermic transition at 38.4 degrees C upon heating and two exothermic transitions between 20 and 30 degrees C upon cooling. These transitions are accompanied by enthalpy changes that are considerably greater than normally observed with typical gel/liquid--crystalline phase transitions and have been assigned to interconversions between lamellar crystalline and lamellar liquid--crystalline forms of this lipid. Both infrared spectroscopy and X-ray diffraction indicate that the lamellar crystalline phase is a highly ordered, substantially dehydrated structure in which the hydrocarbon chains are essentially immobilized in a distorted orthorhombic subcell. Upon heating to temperatures near 38.4 degrees C, this structure converts to a liquid-crystalline phase in which there is excessive swelling of the aqueous interlamellar spaces owing to charge repulsion between, and undulations of, the positively charged lipid surfaces. The polar/apolar interfaces of liquid--crystalline DM-TAP bilayers are not as well hydrated as those formed by other classes of phospho- and glycolipids. Such differences are attributed to the relatively small size of the polar headgroup and its limited capacity for interaction with moieties in the bilayer polar/apolar interface.

Method for obtaining structure and interactions from oriented lipid bilayers.

Precise calculations are made of the scattering intensity I(q) from an oriented stack of lipid bilayers using a realistic model of fluctuations. The quantities of interest include the bilayer bending modulus Kc, the interbilayer interaction modulus B, and bilayer structure through the form factor F(qz). It is shown how Kc and B may be obtained from data at large q(z) where fluctuations dominate. Good estimates of F(qz) can be made over wide ranges of q(z) by using I(q) in q regions away from the peaks and for q(r) not equal0 where details of the scattering domains play little role. Rough estimates of domain sizes can also be made from smaller q(z) data. Results are presented for data taken on fully hydrated, oriented DOPC bilayers in the L(alpha) phase. These results illustrate the advantages of oriented samples compared to powder samples.

Structure of lipid bilayers.

The quantitative experimental uncertainty in the structure of fully hydrated, biologically relevant, fluid (L(alpha)) phase lipid bilayers has been too large to provide a firm base for applications or for comparison with simulations. Many structural methods are reviewed including modern liquid crystallography of lipid bilayers that deals with the fully developed undulation fluctuations that occur in the L(alpha) phase. These fluctuations degrade the higher order diffraction data in a way that, if unrecognized, leads to erroneous conclusions regarding bilayer structure. Diffraction measurements at high instrumental resolution provide a measure of these fluctuations. In addition to providing better structural determination, this opens a new window on interactions between bilayers, so the experimental determination of interbilayer interaction parameters is reviewed briefly. We introduce a new structural correction based on fluctuations that has not been included in any previous studies. Updated measurements, such as for the area compressibility modulus, are used to provide adjustments to many of the literature values of structural quantities. Since the gel (L(beta)') phase is valuable as a stepping stone for obtaining fluid phase results, a brief review is given of the lower temperature phases. The uncertainty in structural results for lipid bilayers is being reduced and best current values are provided for bilayers of five lipids.

Clarification of the ripple phase of lecithin bilayers using fully hydrated, aligned samples.

Aligned samples of lipid bilayers have been fully hydrated from water vapor in a different type of x-ray chamber. Our use of aligned samples resolves issues concerning the ripple phase that were ambiguous from previous powder studies. In particular, our x-ray diffraction data conclusively demonstrate that, on cooling from the L alpha to the P beta' phase, both chiral and racemic samples of dipalmitoyl phosphatidylcholine (DPPC) exhibit phase coexistence of long and short ripples with a ripple wavelength ratio lambda L/lambda S approximately 1.8. Moreover, the long ripple always forms an orthorhombic unit cell (gamma L = 90 degrees), strongly supporting the possibility that these ripples are symmetric. In contrast, gamma S for short ripples was consistently different from 90 degrees, implying asymmetric ripples. We continue to find no evidence that chirality affects the structure of rippled bilayers. The relative thermodynamic stability of the two types of ripples was investigated and a qualitative free energy diagram is given in which the long ripple phase is metastable. Finally, we suggest a kinetic mechanism, involving loss of water, that promotes formation of the metastable long ripple phase for special thermal protocols.

Lipid bilayer structure.

Fluctuations, inherent in flexible and biologically relevant lipid bilayers, make quantitative structure determination challenging. Shortcomings in older methods of structure determination have been realized and new methodologies have been introduced that take fluctuations into account. The large uncertainty in literature values of lipid bilayer structural parameters is being reduced.

Re-analysis of magic angle spinning nuclear magnetic resonance determination of interlamellar waters in lipid bilayer dispersions.

A recent method to obtain the number of water molecules of hydration of multilamellar lipid vesicles using magic angle spinning nuclear magnetic resonance has been re-examined. The previous interpretation divided the water into bulk and interlamellar water and ignored water in defects (lakes) that are intrinsic to multilamellar lipid vesicles; the result was inconsistent with x-ray results for the lipid DOPC. The new interpretation takes advantage of the reduction of lake water with increased spinning and it uses osmotic pressure measurements to determine the loss of interlamellar water. The new result for DOPC from magic angle spinning is consistent with x-ray results.

Polymorphism in myristoylpalmitoylphosphatidylcholine.

This study focuses on the mixed-chain lipid myristoylpalmitoylphosphatidylcholine (MPPC) near full hydration. The lipid, synthesized according to the procedure of (Mason et al., 1981a, has a low degree of acyl chain migration. When MPPC is temperature-jumped (T-jumped) from the L alpha phase (T = 38 degrees C) to T = 20 degrees C or below, a subgel phase forms; this formation takes less than 1 h at a temperature below T = 12 degrees C. The subgel remains stable up to T = 29 degrees C. When MPPC is T-jumped from the L alpha phase to T = 24 degrees C or above, a ripple phase forms with coexisting ripple wavelengths of 240 A and 130 A. In contrast, when MPPC is melted from the subgel phase, the ripple phase is characterized by bilayers having a single ripple wavelength of 130 A. In agreement with earlier studies (Stumpel et al., 1983; Serrallach et al., 1984. Structure and thermotropic properties of mixed-chain phosphatidylcholine bilayer membranes. Biochemistry 23:713-720.), no stable gel phase was observed. Instead, an ill-defined low-angle X-ray pattern is initially observed, which gradually transforms into the subgel phase below 20 degrees C, or into the ripple phase above 24 degrees C. In the wide-angle X-ray diffraction, a single peak is observed, similar to the ripple phase wide-angle pattern, that either persists above 24 degrees C or transforms into a multi-peaked subgel wide-angle pattern below 20 degrees C. The absence of a gel phase can be understood phenomenologically as the relative dominance of the subgel phase in mixed-chain PCs compared to same-chain PCs. The subgel structure and molecular interactions responsible for this comparative behavior are interesting open issues.

Fluid phase structure of EPC and DMPC bilayers.

X-ray diffraction data taken at high instrumental resolution were obtained for EPC and DMPC under various osmotic pressures, primarily at T = 30 degrees C. The headgroup thickness DHH was obtained from relative electron density profiles. By using volumetric results and by comparing to gel phase DPPC we obtain areas AEPCF = 69.4 +/- 1.1 A2 and ADMPCF = 59.7 +/- 0.2 A2. The analysis also gives estimates for the areal compressibility KA. The AF results lead to other structural results regarding membrane thickness and associated waters. Using the recently determined absolute electrons density profile of DPPC, the AF results also lead to absolute electron density profiles and absolute continuous transforms magnitude of F(q) for EPC and DMPC. Limited measurements of temperature dependence show directly that fluctuations increase with increasing temperature and that a small decrease in bending modulus KC accounts for the increased water spacing reported by Simon et al. (1995) Biophys. J. 69, 1473-1483.

Structure and interactions of fully hydrated dioleoylphosphatidylcholine bilayers.

This study focuses on dioleoylphosphatidylcholine (DOPC) bilayers near full hydration. Volumetric data and high-resolution synchrotron x-ray data are used in a method that compares DOPC with well determined gel phase dipalmitoylphosphatidylcholine (DPPC). The key structural quantity obtained is fully hydrated area/lipid A0 = 72.2 +/- 1.1 A2 at 30 degrees C, from which other quantities such as thickness of the bilayer are obtained. Data for samples over osmotic pressures from 0 to 56 atmospheres give an estimate for the area compressibility of KA = 188 dyn/cm. Obtaining the continuous scattering transform and electron density profiles requires correction for liquid crystal fluctuations. Quantitation of these fluctuations opens an experimental window on the fluctuation pressure, the primary repulsive interaction near full hydration. The fluctuation pressure decays exponentially with water spacing, in agreement with analytical results for soft confinement. However, the ratio of decay length lambda(fl) = 5.8 A to hydration pressure decay length lambda = 2.2 A is significantly larger than the value of 2 predicted by analytical theory and close to the ratio obtained in recent simulations. We also obtain the traditional osmotic pressure versus water spacing data. Our analysis of these data shows that estimates of the Hamaker parameter H and the bending modulus Kc are strongly coupled.

DMSO produces a new subgel phase in DPPC: DSC and X-ray diffraction study.

Equilibrium phases and the kinetics of subgel phase transformation of dipalmitoylphosphatidylcholine (DPPC) hydrated with mixtures of dimethylsulfoxide (DMSO)/water have been studied using differential scanning calorimetry (DSC) and X-ray diffraction (XRD). The rate of gel-to-subgel transformation is decreased with a small increase in X, the DMSO/water mole fraction, but then speeds up and becomes faster than in pure water by X = 0.16. The DSC scans show multiple subgel peaks, some of which can be attributed to impacted domain growth. For X greater than 0.10, XRD shows that there is a new, stable subgel phase, S, which also accounts for some of the multiplicity of DSC peaks. Our electron density profiles show that the thickness of the bilayer in the S phase is greater than in the usual C subgel phase. We suggest that the S subgel phase is characterized by different headgroup ordering and smaller chain tilt angle than in the C subgel phase. Electron density profiles show that increasing X decreases the water space between bilayers in all phases, subgel, gel and fluid (L alpha). For X = 0.20, a different gel phase is also observed that may be due to subtle changes in the orientation of chain tilt first observed in partially dehydrated DMPC. The dehydrating effect of DMSO explains the results of a previous study, confirmed in this study, that increasing the concentration of DMSO raises the main transition temperature and eliminates the ripple phase.

Effect of substrate roughness on D spacing supports theoretical resolution of vapor pressure paradox.

The lamellar D spacing has been measured for oriented stacks of lecithin bilayers prepared on a variety of solid substrates and hydrated from the vapor. We find that, when the bilayers are in the L(alpha) phase near 100% relative humidity, the D spacing is consistently larger when the substrate is rougher than when it is smooth. The differences become smaller as the relative humidity is decreased to 80% and negligible differences are seen in the L(beta') phase. Our interpretation is that rough substrates frustrate the bilayer stack energetically, thereby increasing the fluctuations, the fluctuational repulsive forces, and the water spacing compared with stacks on smooth surfaces. This interpretation is consistent with and provides experimental support for a recently proposed theoretical resolution of the vapor pressure paradox.

Structure of gel phase saturated lecithin bilayers: temperature and chain length dependence.

Systematic low-angle and wide-angle x-ray scattering studies have been performed on fully hydrated unoriented multilamamellar vesicles of saturated lecithins with even chain lengths N = 16, 18, 20, 22, and 24 as a function of temperature T in the normal gel (L beta') phase. For all N, the area per chain Ac increases linearly with T with an average slope dAc/dT = 0.027 A2/degree C, and the lamellar D-spacings also increase linearly with an average slope dD/dT = 0.040 A/degree C. At the same T, longer chain length lecithins have more densely packed chains, i.e., smaller Ac's, than shorter chain lengths. The chain packing of longer chain lengths is found to be more distorted from hexagonal packing than that of smaller N, and the distortion epsilon of all N approaches the same value at the respective transition temperatures. The thermal volume expansion of these lipids is accounted for by the expansion in the hydrocarbon chain region. Electron density profiles are constructed using four orders of low-angle lamellar peaks. These show that most of the increase in D with increasing T is due to thickening of the bilayers that is consistent with a decrease in tilt angle theta and with little change in water spacing with either T or N. Because of the opposing effects of temperature on area per chain Ac and tilt angle 0, the area expansivity alpha A is quite small. A qualitative theoretical model based on competing head and chain interactions accounts for our results.

Structure of the ripple phase in lecithin bilayers.

The phases of the x-ray form factors are derived for the ripple (Pbeta') thermodynamic phase in the lecithin bilayer system. By combining these phases with experimental intensity data, the electron density map of the ripple phase of dimyristoyl-phosphatidylcholine is constructed. The phases are derived by fitting the intensity data to two-dimensional electron density models, which are created by convolving an asymmetric triangular ripple profile with a transbilayer electron density profile. The robustness of the model method is indicated by the result that many different models of the transbilayer profile yield essentially the same phases, except for the weaker, purely ripple (0,k) peaks. Even with this residual ambiguity, the ripple profile is well determined, resulting in 19 angstroms for the ripple amplitude and 10 degrees and 26 degrees for the slopes of the major and the minor sides, respectively. Estimates for the bilayer head-head spacings show that the major side of the ripple is consistent with gel-like structure, and the minor side appears to be thinner with lower electron density.

X-ray structure determination of fully hydrated L alpha phase dipalmitoylphosphatidylcholine bilayers.

Bilayer form factors obtained from x-ray scattering data taken with high instrumental resolution are reported for multilamellar vesicles of L alpha phase lipid bilayers of dipalmitoylphosphatidylcholine at 50 degrees C under varying osmotic pressure. Artifacts in the magnitudes of the form factors due to liquid crystalline fluctuations have been eliminated by using modified Caillé theory. The Caillé fluctuation parameter eta 1 increases systematically with increasing lamellar D spacing and this explains why some higher order peaks are unobservable for the larger D spacings. The corrected form factors fall on one smooth continuous transform F(q); this shows that the bilayer does not change shape as D decreases from 67.2 A (fully hydrated) to 60.9 A. The distance between headgroup peaks is obtained from Fourier reconstruction of samples with four orders of diffraction and from electron density models that use 38 independent form factors. By combining these results with previous gel phase results, area AF per lipid molecule and other structural quantities are obtained for the fluid L alpha phase. Comparison with results that we derived from previous neutron diffraction data is excellent, and we conclude from diffraction studies that AF = 62.9 +/- 1.3 A2, which is in excellent agreement with a previous estimate from NMR data.

Anomalous phase behavior of long chain saturated lecithin bilayers.

X-ray scattering has been performed on fully hydrated unoriented multilamellar vesicles of lecithins with even chain lengths n from 16 to 24 as a function of temperature in chain ordered phases. The longer chain lengths, n > or = 20, show anomalous behavior compared to the shorter chain lengths, n < 20. This report concentrates on n = 24. Although the history and time dependence shows that equilibrium was not always achieved, it appears that there is a second gel-like phase G2 below 40 degrees C. The G2 phase has a small tilt angle and opposite hexagonal symmetry breaking from the usual G1 gel phase. Also, as T is raised above 45 degrees C, the wide-angle data suggest the appearance of a phase with hexagonal chain packing and small chain tilt angle.

Small-angle x-ray scattering from lipid bilayers is well described by modified Caillé theory but not by paracrystalline theory.

X-ray scattering data at high instrumental resolution are reported for multilamellar vesicles of L alpha phase lipid bilayers of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine at 50 degrees C under varying osmotic pressure. The data are fitted to two theories that account for noncrystalline disorder, paracrystalline theory (PT) and modified Caillé theory (MCT). The MCT provides good fits to the data, much better than the PT fits. The particularly important characteristic of MCT is the long power law tails in the scattering. PT fits (as well as ordinary integration with no attempt to account for the noncrystalline disorder) increasingly underestimate this scattering intensity as the order h increases, thereby underestimating the form factors used to obtain electron density profiles.

Kinetics of subgel formation in DPPC: X-ray diffraction proves nucleation-growth hypothesis.

Wide-angle and low-angle X-ray diffraction data were obtained during the time course of the gel to subgel phase transformation in fully hydrated DPPC. When the system was kept close to equilibrium by following a T-jump protocol, the X-ray data unequivocally demonstrate the coexistence of growing subgel and shrinking gel domains. When the system was supercooled and held further from equilibrium as in previous studies, the kinetic behavior was more complicated. These data prove that the basic mechanism for the gel to subgel phase transformation is one of nucleation of subgel domains followed by growth of the domains.