Flip-flop Rate Research Articles

Kinetics and Thermodynamics of Flip-Flop in Binary Phospholipid Membranes Measured by Sum-Frequency Vibrational Spectroscopy

In order to better characterize the dependence of lipid flip-flop rate and thermodynamics on the nature of the lipid headgroup, we have studied the kinetics of flip-flop for single-lipid and mixed-lipid bilayers consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) as a function of both pressure and temperature. The kinetics of flipping were studied by sum-frequency vibrational spectroscopy (SFVS), which does not require exogenous chemical labeling of the lipid species of interest. Additionally, SFVS may be employed to track only a single species (DSPE or DSPC) within a binary mixture by selective deuteration of the matrix lipid to make it spectroscopically inactive. Using this approach, we have found the flip-flop of pure DSPE to be slower than the flip-flop of pure DSPC by nearly 2 orders of magnitude. The thermodynamics of the pure systems were analyzed in order to better understand the physical factors underlying their transmembrane dynamics. Headgroup hydrophobicity and associated solvent effects, as well as lipid packing constraints, appear to play a key role in determining the rate of flip-flop for these two species. For mixtures of DSPE + DSPC, both components exhibited similar rates of flip-flop at a given mole fraction of DSPE. The kinetics and thermodynamics of flip-flop in the mixtures did not vary uniformly with changing composition but were well correlated to changes in the molecular packing as a function of DSPE content in the bilayer.

Molecular View of Cholesterol Flip-Flop and Chemical Potential in Different Membrane Environments

The relative stability of cholesterol in cellular membranes and the thermodynamics of fluctuations from equilibrium have important consequences for sterol trafficking and lateral domain formation. We used molecular dynamics computer simulations to investigate the partitioning of cholesterol in a systematic set of lipid bilayers. In addition to atomistic simulations, we undertook a large set of coarse grained simulations, which allowed longer time and length scales to be sampled. Our results agree with recent experiments (Steck, T. L.; et al. Biophys. J. 2002, 83, 2118-2125) that the rate of cholesterol flip-flop can be fast on physiological time scales, while extending our understanding of this process to a range of lipids. We predicted that the rate of flip-flop is strongly dependent on the composition of the bilayer. In polyunsaturated bilayers, cholesterol undergoes flip-flop on a submicrosecond time scale, while flip-flop occurs in the second range in saturated bilayers with high cholesterol content. We also calculated the free energy of cholesterol desorption, which can be equated to the excess chemical potential of cholesterol in the bilayer compared to water. The free energy of cholesterol desorption from a DPPC bilayer is 80 kJ/mol, compared to 67 kJ/mol for a DAPC bilayer. In general, cholesterol prefers more ordered and rigid bilayers and has the lowest affinity for bilayers with two polyunsaturated chains. Overall, the simulations provide a detailed molecular level thermodynamic description of cholesterol interactions with lipid bilayers, of fundamental importance to eukaryotic life.

Mechanism of Membrane Permeabilization by a Bacterial Trehalose Lipid Biosurfactant Produced by Rhodococcus sp.

The interactions of a succinoyl bacterial trehalose lipid biosurfactant produced by Rhodococcus sp. with phospholipid vesicles, leading to membrane permeabilization, are studied by means of calorimetric and fluorescence and absorption spectroscopical techniques in search for a molecular model. The critical micelle concentration (CMC) of trehalose lipid is determined, by surface tension measurements, to be 300 muM. Binding of trehalose lipid to palmitoyloleoylphosphatidylcholine membranes is studied by means of isothermal titration calorimetry. The partition constant, in conjunction with the CMC, indicates that trehalose lipid behaves as a weak detergent, which prefers membrane incorporation over micellization. Addition of trehalose lipid to palmitoyloleoylphosphatidylcholine large unilamellar vesicles results in a size-selective leakage of entrapped solutes to the external medium. Experimental evidence is provided to support the requirement of a stage of flip-flop prior to membrane permeabilization, and the rate of flip-flop is measured using fluorescent probes assays. The lipid composition of the target membrane is found to modulate the leakage process to a great extent. It is proposed that trehalose lipid incorporates into phosphatidylcholine membranes and segregates within lateral domains which may constitute membrane defects or "pores", through which the leakage of small solutes might take place. The results presented here contribute to the knowledge of the molecular mechanisms underlying the membrane-related biological actions of this bacterial trehalose lipid biosurfactant.

Transmembrane cholesterol migration in planar lipid membranes measured with Vibrio cholerae cytolysin as molecular tool

The rate of transbilayer movement (flip-flop) of cholesterol was estimated using planar bilayers with defined initial asymmetry, formed by the opposing monolayers technique. Vibrio cholerae cytolysin (VCC) was utilized as a molecular tool for measuring the cholesterol concentration in the cis leaflet of asymmetric bilayers. To quantify cholesterol flip-flop in planar lipid bilayers, a mathematical model was developed. It considers both the lateral diffusion rate of cholesterol within each monolayer and the flip-flop rate. The difference in initial and steady-state cholesterol contents in bilayer leaflets was used as a start point. Assuming the lateral diffusion coefficient to be of 1 × 10 −8 cm 2 s −1, the characteristic time of cholesterol flip-flop at 25 ± 2 °C was estimated as <10 s.

Cholesterol Flip-flop And Chemical Potential In A Systematic Set Of Lipid Bilayers

Cholesterol is a necessary component of animal cellular membranes. The concentration of cholesterol varies from 0-5 mol% in the endoplasmic reticulum to 25-40mol% in the plasma membrane. Thermal fluctuations cause cholesterol to move normal to the plane of the bilayer. At the extremes, cholesterol can translocate across the bilayer (flip-flop) and diffuse from the bilayer into water (desorption). We have used atomistic and coarse grained molecular dynamics computer simulations to investigate the partitioning of cholesterol through a systematic set of lipid bilayers. Atomistic simulations provide detailed analysis, while inexpensive coarse grained simulations allow more bilayers to be investigated and longer time scales to be sampled. From the coarse grained simulations, cholesterol flip-flop was directly observed, and the rate matched our estimate from the free energy barrier. We find the rate of cholesterol flip-flop is fast and strongly dependent on the structure of the bilayer. The rate of flip-flop is on the microsecond range in fluid, disordered poly-unsaturated bilayers, and on the second range in rigid, ordered bilayers with high cholesterol content. The chemical potential of cholesterol in the bilayer compared to water is equal to our free energies of desorption. We can infer the relative affinity of cholesterol for the bilayers by comparing the chemical potentials. We find cholesterol prefers more ordered and rigid bilayers with saturated acyl tails, and high cholesterol content. Cholesterol has the lowest affinity for poly-unsaturated lipids.

Thermodynamic Analysis of the Effect of Cholesterol on Dipalmitoylphosphatidylcholine Lipid Membranes

Cholesterol is an important component of eukaryotic cellular membranes. Despite extensive literature on the physiochemical effects of cholesterol on membranes, much remains unknown about the precise role of cholesterol and its molecular interactions in membranes. Regular thermal fluctuations of lipids normal to the plane of the membrane are biologically relevant for many processes, such as interactions with enzymes, elastic properties, and hydrophobic matching, while larger fluctuations are involved in vesicle budding and fusion, passive lipid flip-flop, and pore formation. Here we used molecular dynamics simulations to investigate the thermodynamic effect of the cholesterol concentration on dipalmitoylphosphatidylcholine (DPPC) bilayers. We calculated the potentials of mean force for DPPC partitioning in DPPC bilayers containing 20 and 40 mol % cholesterol. Increasing the cholesterol content increases the free energy barrier for transferring the headgroup of DPPC to the center of the bilayer and slows the rate of DPPC flip-flop by orders of magnitude. Cholesterol increases the order, thickness, and rigidity of the bilayers, which restricts bilayer deformations and prevents pore formation. While DPPC flip-flop is pore-mediated in a pure bilayer, we do not observe pores in the 20 and 40 mol % bilayers. Increasing the cholesterol concentration causes a decrease in the free energy to transfer DPPC from its equilibrium position into bulk waterindicating that DPPC prefers to be in cholesterol-free bilayers. We also observe a reduction in small fluctuations of DPPC normal to the bilayer as the cholesterol concentration is increased.

Time-Dependent Interactions of the Two Porphyrinic Compounds Chlorin e6 and Mono-l-aspartyl-chlorin e6 with Phospholipid Vesicles Probed by NMR Spectroscopy

The distribution processes of chlorin e6 (CE) and monoaspartyl-chlorin e6 (MACE) between the outer and inner phospholipid monolayers of 1,2-dioleoyl-phosphatidylcholine (DOPC) vesicles were monitored by 1H NMR spectroscopy through analysis of chemical shifts and line widths of the DOPC vesicle resonances. Chlorin adsorption to the outer vesicle monolayer induced changes in the DOPC 1H NMR spectrum. Most pronounced was a split of the N-methyl choline resonance, allowing for separate analysis of inner and outer vesicle layers. Transbilayer distribution of the chlorin compounds was indicated by time-dependent characteristic spectral changes of the DOPC resonances. Kinetic parameters for the flip-flop processes, that is, half-lives and rate constants, were obtained from the experimental data points. In comparison to CE, MACE transbilayer movement was significantly reduced, with MACE remaining more or less attached to the outer membrane layer. The distribution coefficients for CE and MACE between the vesicular and aqueous phase were determined. Both CE and MACE exhibited a high affinity for the vesicular phase. For CE, a positive correlation was found between transfer rate and increasing molar ratio CE/DOPC. Enhanced membrane rigidity induced by increasing amounts of cholesterol into the model membrane was accompanied by a decrease of CE flip-flop rates across the membrane. The present study shows that the movement of porphyrins across membranes can efficiently be investigated by 1H NMR spectroscopy and that small changes in porphyrin structure can have large effects on membrane kinetics.

Cholesterol Shows Preference for the Interior of Polyunsaturated Lipid Membranes

Recent neutron scattering experiments showed a striking manifestation of the aversion between polyunsaturated fatty acid (PUFA)-containing lipids and cholesterol. Selectively deuterated cholesterol/ 1,2-diarachidonylphosphatidylcholine (DAPC) samples revealed that the hydroxyl of the sterol resides at the center of the bilayer. Here we use a recently parametrized coarse grain simulation model to shed light on these puzzling experimental observations. Using a simulation setup in close correspondence to the experimental conditions, we reproduce the experimental neutron scattering profiles to a large extent. The simulations allow us to analyze the behavior of cholesterol in detail; we show that the interaction of cholesterol with the PUFA chains of DAPC leads to a fast flip-flop rate for the sterol and an increased preference of the sterol for the unusual location embedded between the monolayer leaflets.

Flippase Activity Detected with Unlabeled Lipids by Shape Changes of Giant Unilamellar Vesicles

Transbilayer movement of phospholipids in biological membranes is mediated by energy-dependent and energy-independent flippases. Available methods for detection of flippase mediated transversal flip-flop are essentially based on spin-labeled or fluorescent lipid analogues. Here we demonstrate that shape change of giant unilamellar vesicles (GUVs) can be used as a new tool to study the occurrence and time scale of flippase-mediated transbilayer movement of unlabeled phospholipids. Insertion of lipids into the external leaflet created an area difference between the two leaflets that caused the formation of a bud-like structure. Under conditions of negligible flip-flop, the bud was stable. Upon reconstitution of the energy-independent flippase activity of the yeast endoplasmic reticulum into GUVs, the initial bud formation was reversible, and the shapes were recovered. This can be ascribed to a rapid flip-flop leading to relaxation of the monolayer area difference. Theoretical analysis of kinetics of shape changes provides self-consistent determination of the flip-flop rate and further kinetic parameters. Based on that analysis, the half-time of phospholipid flip-flop in the presence of endoplasmic reticulum proteins was found to be on the order of few minutes. In contrast, GUVs reconstituted with influenza virus protein formed stable buds. The results argue for the presence of specific membrane proteins mediating rapid flip-flop.

Is Membrane Transport of FFA Mediated by Lipid, Protein, or Both?

![Figure][1] J. Patrick Kampf Torrey Pines Institute for Molecular Studies, San Diego, California ![Figure][1] Alan M. Kleinfeld Torrey Pines Institute for Molecular Studies, San Diego, California akleinfeld{at}tpims.org The mechanism of free fatty acid (FFA)

Facile Lipid Flip-Flop in a Phospholipid Bilayer Induced by Gramicidin A Measured by Sum-Frequency Vibrational Spectroscopy

The first direct experimental evidence that gramicidin A (gA), a transmembrane peptide, facilitates the translocation of unlabeled lipids in a phospholipid bilayer was obtained with sum-frequency vibrational spectroscopy (SFVS). SFVS was used to investigate the effect of gA on lipid flip-flop in a planar 1,2-distearoyl- sn-glycero-3-phosphocholine (DSPC) lipid bilayer. The kinetics of lipid translocation were determined by an analysis of the SFVS intensity versus time at different temperatures in the presence of 2 mol % gA. The rate constants of DSPC flip-flop increase from 2 to 10 times relative to the pure DSPC system. The results indicate that facial lipid exchange can be induced by a hydrophobic transmembrane helix. The increase in lipid flip-flop rates is correlated to an increase in the gauche content of the lipid tails. The results suggest that membrane defects induced by the presence of integral membrane proteins may play a large role in modulating the rate of lipid flip-flop.

The role of polyunsaturated lipids in membrane raft function

Docosahexaenoic acid (DHA), with 22 carbons and six double bonds, is the longest, most unsaturated fatty acid commonly found in humans. It represents the extreme example of an omega-3 (n-3) polyunsaturated fatty acid. Since early epidemiology studies, DHA has been linked to alleviation of an enormous number of human afflictions, including heart disease, cancer and neurological disorders. How one simple molecule can affect so many seemingly unrelated abnormalities has been a contentious question for many years. One research direction has investigated events that follow the uptake of DHA into animal cell plasma membrane phospholipids. Summarized here is a variety of membrane properties impacted by the incorporation of DHA. DHA’s dynamic shape, consisting of multiple configurations, is very different from what its static, stick structure would indicate. DHA-containing phospholipids have a wide hydrophobic base compared with their hydrophilic head and so induce nega tive curvature strain that severely impacts the activity of a variety of important membrane proteins. The unusual structure means that DHA-rich membranes are also surprisingly thin and support high permeability, compression, fusion and flip-flop rates. DHA does not exist in an environment that is independent from other membrane lipids. Of particular interest is the interaction of DHA-containing phospholipids with the major lipid raft components cholesterol and sphingomyelin. From a wide variety of biophysical studies, primarily done on model lipid monolayers and bilayers, a new hypothesis is proposed suggesting that DHA may alter plasma membrane lipid raft structure and hence essential cell signaling events. A fundamental role for DHA affecting a feature common to all cells, membrane structure and function, may explain its wide variety of reputed health benefits. Keywords: docosahexaenoic acid; lipid rafts; membrane structure; polyunsaturated fatty acids; n-3

Flip-Flop in Adsorbed Bilayers

There is very rapid exchange between molecules between an adsorbed (supported) bilayer and solution. Complete exchange of a bilayer occurs within seconds; the actual exchange process may be faster but is limited by the resolution of our technique. The exchange process was monitored by infrared spectroscopy, which can independently monitor deuterium-labeled and unlabeled surfactants, and surface selectivity was achieved by using an internal reflection configuration. Total exchange requires exchange of both the inner and outer layer. We demonstrate that the desorption of a monolayer into water is extremely slow, yet exchange of the inner layer into surfactant solution is extremely fast when a bilayer is present. This shows that flip-flop for an adsorbed bilayer is very fast. Rapid flip-flop at interfaces is in sharp contrast to the extremely slow rate of flip-flop in vesicles. We hypothesize that the rapid rate of flip-flop in adsorbed layers is a consequence of surface-induced defects; in free vesicles, the defects are closed by the film pressure.

The mechanism of ABCG5/ABCG8 in biliary cholesterol secretion in mice

The main player in biliary cholesterol secretion is the heterodimeric transporter complex, ABCG5/ABCG8, the function of which is necessary for the majority of sterols secreted into bile. It is not clear whether the primary step in this process is flopping of cholesterol from the inner to the outer leaflet of the canalicular membrane, with desorption by mixed micelles, or decreasing of the activation energy required for cholesterol desorption from the outer membrane leaflet. In this study, we investigated these mechanisms by infusing Abcg8(+/+), Abcg8(+/-), and Abcg8(-/-) mice with hydrophilic and hydrophobic bile salts. In Abcg8(-/-) mice, this failed to substantially stimulate biliary cholesterol secretion. Infusion of the hydrophobic bile salt taurodeoxycholate also resulted in cholestasis, which was induced in Abcg8(-/-) mice at a much lower infusion rate compared with Abc8(-/-) and Abcg8(+/-) mice, suggesting a reduced cholesterol content in the outer leaflet of the canalicular membrane. Indeed, isolation of canalicular membranes revealed a reduction of 45% in cholesterol content under these conditions in Abcg8(-/-) mice. Our data support the model that ABCG5/ABCG8 primarily play a role in flopping cholesterol (and sterols) from the inner leaflet to the outer leaflet of the canalicular membrane.

Lipids Out of Equilibrium: Energetics of Desorption and Pore Mediated Flip-Flop

The potential of mean force (PMF) of a phospholipid in a bilayer is a key thermodynamic property that describes the energetic cost of localized lipid defects. We have calculated the PMF by umbrella sampling using molecular dynamics simulations. The profile has a deep minimum at the equilibrium position in the bilayer and steeply rises for displacements both deeper into the bilayer and moving away from the bilayer. As the lipid loses contact with the bilayer, the profile abruptly flattens without a significant barrier. The calculated free energy difference of 80 kJ/mol between the minimum of the PMF and the value in water agrees well with the free energy difference calculated from the experimentally measured critical micelle concentration. Significant water/lipid defects form when a lipid is forced into the bilayer interior, in the form of a small water pore that spans the membrane. The energy required to form such a water pore is also found to be 80 kJ/mol. On the basis of this energy, we estimate the lipid flip-flop rate and permeability rate of sodium ions. The resulting rates are in good agreement with experimental measurements, suggesting lipid flip-flop and basal permeability of ions are pore mediated.

How fatty acids of different chain length enter and leave cells by free diffusion

Opposing views exist as to how unesterified fatty acids (FA) enter and leave cells. It is commonly believed that for short- and medium-chain FA free diffusion suffices whereas it is questioned whether proteins are required to facilitate transport of long-chain fatty acid (LCFA). Furthermore, it is unclear whether these proteins facilitate binding to the plasma membrane, trans-membrane movement, dissociation into the cytosol and/or transport in the cytosol. In this mini-review we approach the controversy from a different point of view by focusing on the membrane permeability constant ( P) of FA with different chain length. We compare experimentally derived values of the P of short and medium-chain FA with values of apparent permeability coefficients for LCFA calculated from their dissociation rate constant ( k off), flip-flop rate constant ( k flip) and partition coefficient ( K p) in phospholipid bilayers. It was found that Overton's rule is valid as long as k flip≪ k off. With increasing chain length, the permeability increases according to increasing K p and reaches a maximum for LCFA with chain length of 18 carbons or longer. For fast flip-flop (e.g. k flip=15 s −1), the apparent permeability constant for palmitic acid is very high ( P app=1.61 cm/s). Even for a slow flip-flop rate constant (e.g. k flip=0.3 s −1), the permeability constant of LCFA is still several orders of magnitude larger than the P of water and other small non-electrolytes. Since polyunsaturated FA have basically the same physico-chemical properties as LCFA, they have similar membrane permeabilities. The implications for theories involving proteins to facilitate uptake of FA are discussed.

Different Mechanisms of Free Fatty Acid Flip-Flop and Dissociation Revealed by Temperature and Molecular Species Dependence of Transport across Lipid Vesicles

The mechanism of free fatty acid (FFA) transport across membranes is a subject of intense investigation. We have demonstrated recently that flip-flop is the rate-limiting step for transport of oleic acid across phospholipid vesicles (Cupp, D., Kampf, J. P., and Kleinfeld, A. M. (2004) Biochemistry 43, 4473-4481). To better understand the nature of the flip-flop barrier, we measured the temperature dependence of a series of saturated and monounsaturated FFA. We determined the rate constants for flip-flop and dissociation for small (SUV), large (LUV), and giant (GUV) unilamellar vesicles composed of egg phosphatidylcholine. For all FFA and vesicle types, dissociation was faster than flip-flop, and for all FFA, flip-flop and dissociation were faster in SUV than in LUV or GUV. Rate constants for both flip-flop and dissociation decreased exponentially with increasing FFA size. However, only the flip-flop rate constants increased significantly with temperature; the barrier to flip-flop was virtually entirely due to an enthalpic activation free energy. The barrier to dissociation was primarily entropic. Analysis in terms of a simple free volume (V(f)) model revealed V(f) values for flip-flop that ranged between approximately 12 and 15 Angstroms(3), with larger values for SUV than for LUV or GUV. V(f) values increased with temperature, and this temperature dependence generated the enthalpic barrier to flip-flop. The barrier for dissociation and its size dependence primarily reflect the aqueous solubility of FFA. These are the first results to distinguish the energetics of flipflop and dissociation. This should lead to a better understanding of the mechanisms governing FFA transport across biological membranes.

The role of polyunsaturated lipids in membrane raft function

Docosahexaenoic acid (DHA), with 22 carbons and six double bonds, is the longest, most unsaturated fatty acid commonly found in humans. It represents the extreme example of an omega-3 (n-3) polyunsaturated fatty acid. Since early epidemiology studies, DHA has been linked to alleviation of an enormous number of human afflictions, including heart disease, cancer and neurological disorders. How one simple molecule can affect so many seemingly unrelated abnormalities has been a contentious question for many years. One research direction has investigated events that follow the uptake of DHA into animal cell plasma membrane phospholipids. Summarized here is a variety of membrane properties impacted by the incorporation of DHA. DHA’s dynamic shape, consisting of multiple configurations, is very different from what its static, stick structure would indicate. DHA-containing phospholipids have a wide hydrophobic base compared with their hydrophilic head and so induce nega tive curvature strain that severely impacts the activity of a variety of important membrane proteins. The unusual structure means that DHA-rich membranes are also surprisingly thin and support high permeability, compression, fusion and flip-flop rates. DHA does not exist in an environment that is independent from other membrane lipids. Of particular interest is the interaction of DHA-containing phospholipids with the major lipid raft components cholesterol and sphingomyelin. From a wide variety of biophysical studies, primarily done on model lipid monolayers and bilayers, a new hypothesis is proposed suggesting that DHA may alter plasma membrane lipid raft structure and hence essential cell signaling events. A fundamental role for DHA affecting a feature common to all cells, membrane structure and function, may explain its wide variety of reputed health benefits. Keywords: docosahexaenoic acid; lipid rafts; membrane structure; polyunsaturated fatty acids; n-3

Thermodynamics of Lipid Membrane Solubilization by Sodium Dodecyl Sulfate

We provide a comprehensive thermodynamic description of lipid membrane dissolution by a charged detergent. To this end, we have studied the interactions between the anionic detergent sodium dodecyl sulfate (SDS) and the zwitterionic phospholipid 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC) in dilute aqueous solution (10 mM phosphate buffer, 154 mM NaCl, pH 7.4). Thermodynamic parameters of vesicle solubilization and reconstitution, membrane partitioning, and micelle formation were assessed by right-angle light scattering and isothermal titration calorimetry. Membrane translocation and dissolution proceed very slowly at 25°C but are considerably accelerated at 65°C. At this temperature, a simple SDS/POPC phase diagram (comprising vesicular, coexistence, and micellar ranges) and a complete set of partition coefficients and transfer enthalpies were obtained. Electrostatic repulsion effects at the membrane surface were implemented by combining Gouy-Chapman theory with a Langmuir adsorption isotherm to account for Na + binding to membrane-incorporated DS −. This approach offered a quantitative understanding of solubilization and reconstitution processes, which were interpreted in terms of partition equilibria between and ideal mixing in all phases. More than any other property, the transbilayer flip-flop rate under given experimental conditions hence appears to dictate a detergent's suitability for thermodynamically controlled lipid membrane solubilization and reconstitution.

Solvent-free model for self-assembling fluid bilayer membranes: Stabilization of the fluid phase based on broad attractive tail potentials

We present a simple and highly adaptable method for simulating coarse-grained lipid membranes without explicit solvent. Lipids are represented by one head bead and two tail beads, with the interaction between tails being of key importance in stabilizing the fluid phase. Two such tail-tail potentials were tested, with the important feature in both cases being a variable range of attraction. We examined phase diagrams of this range versus temperature for both functional forms of the tail-tail attraction and found that a certain threshold attractive width was required to stabilize the fluid phase. Within the fluid-phase region we find that material properties such as area per lipid, orientational order, diffusion constant, interleaflet flip-flop rate, and bilayer stiffness all depend strongly and monotonically on the attractive width. For three particular values of the potential width we investigate the transition between gel and fluid phases via heating or cooling and find that this transition is discontinuous with considerable hysteresis. We also investigated the stretching of a bilayer to eventually form a pore and found excellent agreement with recent analytic theory.