Steric repulsion between phosphatidylcholine bilayers

The change in pressure needed to bring egg phosphatidylcholine bilayers into contact from their equilibrium separation in excess water has been determined as a function of both distance between the bilayers and water content. A distinct upward break in the pressure-distance relation appears at an interbilayer separation of about 5 A, whereas no such deviation is present in the pressure-water content relation. Thus, this break is not a property of the dehydration process per se, but instead is attributed to steric repulsion between the mobile lipid head groups that extend 2-3 A into the fluid space between bilayers. That is, electron density profiles of these bilayers indicate that the observed break in the pressure-spacing relation occurs at a bilayer separation where extended head groups from apposing bilayers come into steric hindrance. The pressure-spacing data are used to separate steric pressure from the repulsive hydration pressure, as well as to quantitate the range and magnitude of the steric interaction. An appreciable fraction of the measured steric energy can be ascribed to a decrease in configurational entropy due to restricted head-group motion as adjacent bilayers come together.     

Hydration force and bilayer deformation: a reevaluation

The hydration repulsive force between lipid bilayers and the deformability of both gel and liquid-crystalline bilayers have been quantitated by an X-ray diffraction analysis of osmotically stressed liposomes. Both sampling theorem reconstructions and electron density distributions were calculated from diffraction data obtained from multilayers with applied osmotic pressures of 0-50 atm. The bilayer thickness and area per lipid molecule remain nearly constant (to within about 4%) in this pressure range, as adjacent bilayers move from their equilibrium separation in excess water to within 2-4 A of each other. This analysis indicates that the bilayers are relatively incompressible. This results differs from previously published X-ray diffraction studies of bilayer compressibility but agrees with direct mechanical measurements of the bilayer compressibility modulus. It is also found that the hydration repulsive force decays exponentially with separation between bilayers with a decay constant of 1.4 A for gel-state dipalmitoylphosphatidylcholine and 1.7 A for liquid-crystalline egg phosphatidylcholine bilayers. This implies that the exponential decay constant is not necessarily equal to the diameter of a water molecule, as has been previously suggested on experimental and theoretical grounds.     

Direct measurement of the interaction between phosphatidylglycerol bilayers in aqueous electrolyte solutions.

Results are presented of force measurements between deposited bilayers of dimyristoylphosphatidyl glycerol (DMPG) at T greater than Tm, and distearoylphosphatidyl glycerol (DSPG) at T less than Tm. Below a bilayer separation of 100 nm, a repulsive double-layer force is measured, which can be explained through the combined screening and binding effect of the counterions in electrolyte solutions of NaCl, HCl, CaCl2, or mixtures of these. The binding of cations to bilayers in the fluid phase (DMPG) appears to be greater than to bilayers in the gel phase (DSPG). At shorter range, below approximately 3 nm, an attractive interaction is measured in solutions containing CaCl2, which was found to be slightly stronger than the theoretically expected van der Waals interaction. No hydration force was observed to exist in solutions containing CaCl2. In NaCl solutions, the measured interbilayer force can completely be accounted for by the electrostatic repulsion, down to a bilayer separation of at least 2 nm, below which no accurate measurements were possible anymore. Parallel measurements on PG monolayers show that the contraction of a DMPG monolayer following addition of CaCl2 is significantly greater than what is predicted from the change in the double-layer free energy alone. This indicates that changes in the lateral interactions between the lipid headgroups probably involve Ca2+-bridge binding and/or a possible dehydration of the lipid headgroups through Ca2+ binding. The results shed new light on both the interbilayer and intrabilayer interactions of PG and identify the possible factors responsible for the morphological behavior of PG aggregates.     

Binding of divalent cations of dipalmitoylphosphatidylcholine bilayers and its effect on bilayer interaction

We have confirmed that CaCl2 swells the multilayer lattice formed by dipalmitolyphosphatidylcholine (DPPC) in an aqueous solution. Specifically, at room temperature 1 mM CaCl2 causes these lipid bilayers to increase their separation, dw, from 19 A in pure water to greater than 90 A. CaCl2 concentrations greater than 4 mM cause less swelling. We have measured the net repulsive force between the bilayers in 30 mM CaCl2 at T = 25 degrees C (below the acyl chain freezing temperature). For interbilayer separations between 30 and 90 A, the dominant repulsion between bilayers is probably electrostatic; Ca2+ binds to DPPc lecithin bilayers, imparting a charge to them. The addition of NaCl to CaCl2 solutions decreases this repulsion. For dw less than 20 A, the bilayer repulsion appears to be dominated by the "hydration forces" observed previously between both neutral and charged phospholipids. From the electrostatic repulsive force, we estimate the extent of Ca2+ binding to the bilayer surface. The desorption and bound Ca2+, apparent when bilayers are pushed together, is more rapid than one would expect if an association constant governed Ca2+ binding. The association affinity does not appear to be a fixed quantity but rather a sensitive function of ionic strength and bilayer separation.     

Neutron and X-ray diffraction structural analysis of phosphatidylinositol bilayers

Phosphatidylinositol (PI) bilayers, squeezed together by applied osmotic pressures, were studied by both neutron diffraction and X-ray diffraction. The lamellar repeat period for PI bilayers decreased from 9.5 nm at an applied pressure of 1.1.10(6) dyn/cm2 (1.1 atm) to 5.4 nm at an applied pressure of 1.6.10(7) dyn/cm2 (16 atm). Further increases in applied pressure, up to 2.7.10(9) dyn/cm2 (2700 atm) reduced the repeat period by only about 0.3 nm, to 5.1 nm. Thus, a plot of applied pressure versus repeat period shows a sharp upward break for repeat periods less than about 5.4 nm. For repeat periods of less than 5.4 nm, analysis of neutron-scattering density profiles and electron-density profiles indicates that the structure of the PI bilayers changes as the bilayers are dehydrated, even though there are only small changes in the repeat period. These structural changes are most likely due to removal of water from the headgroup regions of the bilayer. D2O/H2O exchange experiments show that, at an applied pressure of 2.8.10(7) dyn/cm2, water is located between adjacent PI headgroups in the plane of the bilayer. We conclude that, although electrostatics provide the dominant long-range repulsive interaction, hydration repulsion and steric hindrance between PI headgroups from apposing bilayers provide the major barriers for the close approach of adjacent PI bilayers for repeat periods less than 5.4 nm. This structural analysis also indicates that the phosphoinositol group extends from the plane of the bilayer into the fluid space between adjacent bilayers. This extended orientation for the headgroup is consistent with electrophoretic measurements on PI vesicles.     

Investigation of temperature-induced phase transitions in DOPC and DPPC phospholipid bilayers using temperature-controlled scanning force microscopy

Under physiological conditions, multicomponent biological membranes undergo structural changes which help define how the membrane functions. An understanding of biomembrane structure-function relations can be based on knowledge of the physical and chemical properties of pure phospholipid bilayers. Here, we have investigated phase transitions in dipalmitoylphosphatidylcholine (DPPC) and dioleoylphosphatidylcholine (DOPC) bilayers. We demonstrated the existence of several phase transitions in DPPC and DOPC mica-supported bilayers by both atomic force microscopy imaging and force measurements. Supported DPPC bilayers show a broad L(beta)-L(alpha) transition. In addition to the main transition we observed structural changes both above and below main transition temperature, which include increase in bilayer coverage and changes in bilayer height. Force measurements provide valuable information on bilayer thickness and phase transitions and are in good agreement with atomic force microscopy imaging data. A De Gennes model was used to characterize the repulsive steric forces as the origin of supported bilayer elastic properties. Both electrostatic and steric forces contribute to the repulsive part of the force plot.     

First-Leaflet Phase Effect on Properties of Phospholipid Bilayer Formed Through Vesicle Adsorption on LB Monolayer

Phospholipid bilayers were formed on mica using the Langmuir–Blodgett technique and liposome fusion, as a model system for biomembranes. Nanometer-scale surface physical properties of the bilayers were quantitatively characterized upon the different phases of the first leaflets. Lower hydration/steric forces on the bilayers were observed at the liquid phase of the first leaflet than at the solid phase. The forces appear to be related to the low mechanical stability of the lipid bilayer, which was affected by the first leaflet phase. The first leaflet phase also influenced the long-range repulsive forces over the second leaflet. Surface forces, measured using a modified probe with an atomic force microscope, showed that lower long-range repulsive forces were also found at the liquid phase of the first leaflet. Force measurements were performed at 300 mM sodium chloride solution so that the effect of the phase on the long-range repulsive forces could be investigated by reducing the effect of the repulsion between the second-leaflet lipid headgroups on the long-range repulsive forces. Forces were analyzed using the Derjaguin–Landau–Verwey–Overbeek theory so that the surface potential and surface charge density of the lipid bilayers were quantitatively acquired for each phase of the first leaflet.     

Variation in hydration forces between neutral phospholipid bilayers: evidence for hydration attraction

It is now generally recognized that hydration forces dominate close interactions of lipid hydrophilic surfaces. The commonality of their characteristics has been reasonably established. However, differences in measured net repulsion, particularly evident when phosphatidylethanolamine (PE) and phosphatidylcholine (PC) bilayers are compared, suggest there exists a variety of behavior wider than expected from earlier models of hydration and fluctuation repulsion balanced by van der Waals attraction. To find a basis for this diverse behavior, we have looked more closely at measured structural parameters, degrees of hydration, and interbilayer repulsive forces for the lamellar phases of the following lipids: 1-palmitoyl-2-oleoyl-PE (POPE), egg PE, transphosphatidylated egg PE (egg PE-T), mono- and dimethylated egg PE-T (MMPE and DMPE), 1-stearoyl-2-oleoyl-PC (SOPC), and mixtures of POPE and SOPC. POPE and SOPC bilayers differ not only in their maximum degrees of hydration but also in the empirical hydration force coefficients and decay lengths that characterize their interaction. When mixed with POPE, SOPC effects sudden and disproportionate increases in hydration. POPE, egg PE, and egg PE-T differ in their degree of hydration, molecular area, and hydration repulsion. A single methylation of egg PE-T almost completely converts its hydration and bilayer repulsive properties to those of egg PC; little progression of hydration is seen with successive methylations. In order to reconcile these observations with the conventional scheme of balancing interbilayer hydration and fluctuation-enhanced repulsion with van der Waals attraction, it is necessary to relinquish the fundamental idea that the decay of hydration forces is a constant determined by the properties of the aqueous medium. Alternatively, one can retain that fundamental idea if one recognizes the possibility that polar group hydration has an attractive component to it. In the latter view, that attractive component originates from interbilayer hydrogen-bonded water bridges between apposing bilayer surfaces, arising from correlation of zwitterionic or other complementary polar groups or from factors that affect polar group solubility. The same Marcelja and Radic formalism that accounts so well for the repulsive component also leads to an estimate of the attractive one. We suggest that the full range of degrees of hydration and of interbilayer spacings observed for different neutral bilayers results in part from variable contributions of the attractive and repulsive hydration components.(ABSTRACT TRUNCATED AT 400 WORDS)     

Direct imaging of individual intrinsic hydration layers on lipid bilayers at Angstrom resolution

The interactions between water and biological molecules have the potential to influence the structure, dynamics, and function of biological systems, hence the importance of revealing the nature of these interactions in relation to the local biochemical environment. We have investigated the structuring of water at the interface of supported dipalmitoylphosphatidylcholine bilayers in the gel phase in phosphate buffer solution using frequency modulation atomic force microscopy (FM-AFM). We present experimental results supporting the existence of intrinsic (i.e., surface-induced) hydration layers adjacent to the bilayer. The force versus distance curves measured between the bilayer and the AFM tip show oscillatory force profiles with a peak spacing of 0.28 nm, indicative of the existence of up to two hydration layers next to the membrane surface. These oscillatory force profiles reveal the molecular-scale origin of the hydration force that has been observed between two apposing lipid bilayers. Furthermore, FM-AFM imaging at the water/lipid interface visualizes individual hydration layers in three dimensions, with molecular-scale corrugations corresponding to the lipid headgroups. The results demonstrate that the intrinsic hydration layers are stable enough to present multiple energy barriers to approaching nanoscale objects, such as proteins and solvated ions, and are expected to affect membrane permeability and transport.     

Direct measurements of forces between phosphatidylcholine and phosphatidylethanolamine bilayers in aqueous electrolyte solutions

We report direct measurements of the full interbilayer force laws (force vs. distance) between bilayers of various phosphatidylcholines and phosphatidylethanolamine in aqueous solutions. Bilayers were first deposited on molecularly smooth (mica) surfaces and the interbilayer forces then measured at a resolution of 1 A. Three types of forces were identified: attractive van der Waals forces, repulsive electrostatic (double-layer) forces, and (at short range) repulsive steric hydration forces. Double-layer forces, which arise from ion binding, were insignificant in monovalent salt solutions, e.g., NaCl up to 1 M, but were already present in solutions containing millimolar levels of CaCl2 and MgCl2, giving rise to forces in excellent agreement with theory. Ca2+ binds more strongly than Mg2+, and both bind less to lecithin bilayers in the fluid state (T greater than Tc). The plane of charge coincides with the location of the negative phosphate groups, while the effective plane of origin of the van der Waals force is 4-5 A farther out. In water, the adhesion energies are in the range 0.10-0.15 erg/cm2 for lecithins and approximately 0.8 erg/cm2 for phosphatidylethanolamine. The adhesion energies vary on addition of salt due to changes in the repulsive double-layer and hydration forces rather than to a change in the attractive van der Waals force. The short-range repulsive forces which balance the van der Waals force at separations of 10-30 A are due to a combination of hydration and steric repulsions, the latter arising from thermal motions of head groups and thickness fluctuations of fluid bilayers (above Tc). It is also concluded that bilayer fusion is not simply related to the interbilayer force law.     

Diffusion of dihydropyridine calcium channel antagonists in cardiac sarcolemmal lipid multibilayers.

A membrane bilayer pathway model has been proposed for the interaction of dihydropyridine (DHP) calcium channel antagonists with receptors in cardiac sarcolemma (Rhodes, D.G., J.G. Sarmiento, and L.G. Herbette. 1985. Mol. Pharmacol. 27:612-623) involving drug partition into the bilayer with subsequent receptor binding mediated (though probably not rate-limited) by diffusion within the bilayer. Recently, we have characterized the partition step, demonstrating that DHPs reside, on a time-average basis, near the bilayer hydrocarbon core/water interface. Drug distribution about this interface may define a plane of local concentration for lateral diffusion within the membrane. The studies presented herein examine the diffusional dynamics of an active rhodamine-labeled DHP and a fluorescent phospholipid analogue (DiIC16) in pure cardiac sarcolemmal lipid multibilayer preparations as a function of bilayer hydration. At maximal bilayer hydration, the drug diffuses over macroscopic distances within the bilayer at a rate identical to that of DiI (D = 3.8 X 10(-8) cm2/s), demonstrating the overall feasibility of the membrane diffusion model. The diffusion coefficients for both drug and lipid decreased substantially as the bilayers were dehydrated. While identical at maximal hydration, drug diffusion was significantly slower than that of DiIC16 in partially dehydrated bilayers, probably reflecting differences in mass distribution of these probes in the bilayer.     

Temperature dependence of the repulsive pressure between phosphatidylcholine bilayers.

Bilayer structure and interbilayer repulsive pressure were measured from 5 to 50 degrees C by the osmotic stress/x-ray diffraction method for both gel and liquid crystalline phase lipid bilayers. For gel phase dibehenoylphosphatidylcholine (DBPC) the bilayer thickness and pressure-distance relations were nearly temperature-independent, and at full hydration the equilibrium fluid spacing increased approximately 1 A, from 10 A at 5 degrees C to 11 A at 50 degrees C. In contrast, for liquid crystalline phase egg phosphatidylcholine (EPC), the bilayer thickness, equilibrium fluid spacing, and pressure-distance relation were all markedly temperature-dependent. As the temperature was increased from 5 to 50 degrees C the EPC bilayer thickness decreased approximately 4 A, and the equilibrium fluid spacing increased from 14 to 21 A. Over this temperature range there was little change in the pressure-distance relation for fluid spacings less than approximately 10 A, but a substantial increase in the total pressure for fluid spacings greater than 10 A. These data show that for both gel and liquid crystalline bilayers there is a short-range repulsive pressure that is nearly temperature-independent, whereas for liquid crystalline bilayers there is also a longer-range pressure that increases with temperature. From analysis of the energetics of dehydration we argue that the temperature-independent short-range pressure is consistent with a hydration pressure due to polarization or electrostriction of water molecules by the phosphorylcholine moiety. For the liquid crystalline phase, the 7 A increase in equilibrium fluid spacing with increasing temperature can be predicted by an increase in the undulation pressure as a consequence of a temperature-dependent decrease in bilayer bending modulus.     

Repulsive interactions between uncharged bilayers. Hydration and fluctuation pressures for monoglycerides.

Pressure versus distance relations have been obtained for solid (gel) and neat (liquid-crystalline) phase uncharged lipid bilayers by the use of x-ray diffraction analysis of osmotically stressed monoglyceride aqueous dispersions and multilayers. For solid phase monoelaidin bilayers, the interbilayer repulsive pressure decays exponentially from a bilayer separation of approximately 7 A at an applied pressure of 3 x 10(7) dyn/cm2 to a separation of approximately 11 A at zero applied pressure, where an excess water phase forms. The decay length is approximately 1.3 A, which is similar to the value previously measured for gel phase phosphatidylcholine bilayers. This implies that the decay length of the hydration pressure does not depend critically on the presence of zwitterionic head groups in the bilayer surface. For liquid-crystalline monocaprylin, the repulsive pressure versus distance curve has two distinct regions. In the first region, for bilayer separations of approximately 3-8 A and applied pressures of 3 x 10(8) to 4 x 10(6) dyn/cm2, the pressure decays exponentially with a decay length of approximately 1.3 A. In the second region, for bilayer separations of approximately 8-22 A and applied pressures of 4 x 10(6) to 1 x 10(5) dyn/cm2, the pressure decays much more gradually and is inversely proportional to the cube of the distance between bilayers. These data imply that two repulsive pressures operate between liquid-crystalline monocaprylin bilayers, the hydration pressure, which dominates at small (3-8 A) bilayer separations, and the fluctuation pressure, which dominates at larger bilayer separations (greater than 8 A) and strongly influences the hydration properties of the liquid-crystalline bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions between charged, uncharged, and zwitterionic bilayers containing phosphatidylglycerol.

Pressure vs. distance relationships have been obtained for phosphatidylglycerol bilayers, in both charged and uncharged states. Water was removed from the lipid multilayers by the application of osmotic pressures in the range of 0-2.7 x 10(9) dyn/cm2, and the distance between adjacent bilayers was obtained from Fourier analysis of lamellar x-ray diffraction data. For phosphatidylglycerol bilayers made electrically neutral either by lowering the pH or by adding equimolar concentrations of the positively charged lipid stearylamine, the pressure-distance data could be fit with a single exponential. The measured decay lengths were 1.1 A at low pH and 1.5 A with stearylamine, which are similar to decay lengths of the hydration pressure found for gel phases of other neutral bilayers. In addition, the magnitude of this repulsive pressure was proportional to the square of the Volta potential (equivalent to the dipole potential for electrically neutral bilayers) measured in monolayers in equilibrium with bilayers, in agreement with results previously found for the hydration pressure between phosphatidylcholine bilayers. For charged phosphatidylglycerol bilayers, the pressure-distance relation had two distinct regions. For bilayer separations greater than 10 A, the pressure-distance data had an exponential decay length (11 A) and a magnitude consistent with that expected for electrostatic repulsion from double-layer theory. For bilayer separations of 2-10 A, the pressure decayed much more rapidly with increasing bilayer separation (decay length less than 1 A). We interpret these data at low bilayer separations in terms of a combination of hydration repulsion and steric hindrance between the lipid head groups and the sodium ions trapped between apposing bilayers.     

Membrane organization in soybean seeds during hydration

The ability of seeds to withstand dehydration indicates that their membranes may maintain structural integrity even when dry. Analysis of polar lipids (the principal lipidic constituents of the membranes) from soybean seeds (Glycine-max (L.) Merr.) by X-ray diffraction indicated that even in the dehydrated state the lipids retained a lamellar (bilayer) configuration. As the degree of hydration was raised, evidence of some structural alteration (apparent as an abrupt increase in bilayer spacing) was obtained from diffraction patterns of both the extracted lipid and particles of seed tissue. In seed tissue this increase in bilayer spacing occurred at a hydration level just above that at which free water could be detected by nuclear-magnetic-resonance analysis. The water content at which the increase in bilayer spacing occurred was higher in the seed tissue than in the extracted polar lipids, probably because other cell components restricted the availability of free water in the seed.Abbreviation NMR nuclear-magnetic resonance     

Solid-State H NMR Shows Equivalence of Dehydration and Osmotic Pressures in Lipid Membrane Deformation

Lipid bilayers represent a fascinating class of biomaterials whose properties are altered by changes in pressure or temperature. Functions of cellular membranes can be affected by nonspecific lipid-protein interactions that depend on bilayer material properties. Here we address the changes in lipid bilayer structure induced by external pressure. Solid-state ²H NMR spectroscopy of phospholipid bilayers under osmotic stress allows structural fluctuations and deformation of membranes to be investigated. We highlight the results from NMR experiments utilizing pressure-based force techniques that control membrane structure and tension. Our ²H NMR results using both dehydration pressure (low water activity) and osmotic pressure (poly(ethylene glycol) as osmolyte) show that the segmental order parameters (S_CD) of DMPC approach very large values of ≈0.35 in the liquid-crystalline state. The two stresses are thermodynamically equivalent, because the change in chemical potential when transferring water from the interlamellar space to the bulk water phase corresponds to the induced pressure. This theoretical equivalence is experimentally revealed by considering the solid-state ²H NMR spectrometer as a virtual osmometer. Moreover, we extend this approach to include the correspondence between osmotic pressure and hydrostatic pressure. Our results establish the magnitude of the pressures that lead to significant bilayer deformation including changes in area per lipid and volumetric bilayer thickness. We find that appreciable bilayer structural changes occur with osmotic pressures in the range of 10−100 atm or lower. This research demonstrates the applicability of solid-state ²H NMR spectroscopy together with bilayer stress techniques for investigating the mechanism of pressure sensitivity of membrane proteins.     

Cholesterol modifies the short-range repulsive interactions between phosphatidylcholine membranes

Pressure versus distance relationships have been obtained for egg phosphatidylcholine bilayers containing a range of cholesterol concentrations. Water was removed from between adjacent bilayers by the application of osmotic pressures in the range of 0.4-2600 atm (4 x 10(5)-2.6 x 10(9) dyn/cm2), and the distance between adjacent bilayers was obtained by Fourier analysis of X-ray diffraction data. For applied pressures up to about 50 atm and bilayer surface separations of 15-5 A, the incorporation of up to equimolar cholesterol has little influence on plots of pressure versus bilayer separation. However, for the higher applied pressures, cholesterol reduces the interbilayer separation distance by an amount that depends on the cholesterol concentration in the bilayer. For example, the incorporation of equimolar cholesterol reduces the distance between bilayers by as much as 6 A at an applied pressure of 2600 atm. At this applied pressure, electron density profiles show that the high-density head-group peaks from apposing bilayers have merged. This indicates that equimolar concentrations of cholesterol spread the lipid molecules apart in the plane of the bilayer enough to allow the phosphatidylcholine head groups from apposing bilayers to interpenetrate as the bilayers are squeezed together. All of these X-ray and pressure-distance data indicate that, by reducing the volume fraction of phospholipid head groups, cholesterol markedly reduces the steric repulsion between apposing bilayers but has a much smaller effect on the sum of the longer ranged repulsive hydration and fluctuation pressures. Increasing concentrations of cholesterol monotonically increase the dipole potential of egg phosphatidylcholine monolayers, from 415 mV with no cholesterol to 493 mV with equimolar cholesterol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solid-state ²H NMR shows equivalence of dehydration and osmotic pressures in lipid membrane deformation

Lipid bilayers represent a fascinating class of biomaterials whose properties are altered by changes in pressure or temperature. Functions of cellular membranes can be affected by nonspecific lipid-protein interactions that depend on bilayer material properties. Here we address the changes in lipid bilayer structure induced by external pressure. Solid-state 2H NMR spectroscopy of phospholipid bilayers under osmotic stress allows structural fluctuations and deformation of membranes to be investigated. We highlight the results from NMR experiments utilizing pressure-based force techniques that control membrane structure and tension. Our 2H NMR results using both dehydration pressure (low water activity) and osmotic pressure (poly(ethylene glycol) as osmolyte) show that the segmental order parameters (S(CD)) of DMPC approach very large values of ≈ 0.35 in the liquid-crystalline state. The two stresses are thermodynamically equivalent, because the change in chemical potential when transferring water from the interlamellar space to the bulk water phase corresponds to the induced pressure. This theoretical equivalence is experimentally revealed by considering the solid-state 2H NMR spectrometer as a virtual osmometer. Moreover, we extend this approach to include the correspondence between osmotic pressure and hydrostatic pressure. Our results establish the magnitude of the pressures that lead to significant bilayer deformation including changes in area per lipid and volumetric bilayer thickness. We find that appreciable bilayer structural changes occur with osmotic pressures in the range of 10-100 atm or lower. This research demonstrates the applicability of solid-state 2H NMR spectroscopy together with bilayer stress techniques for investigating the mechanism of pressure sensitivity of membrane proteins.     

Protein shape change has a major effect on the gating energy of a mechanosensitive channel

Increasing experimental evidence has shown that membrane protein functionality depends on molecular composition of cell membranes. However, the origin of this dependence is not fully understood. It is reasonable to assume that specific lipid-protein interactions are important, yet more generic effects due to mechanical properties of lipid bilayers likely play a significant role too. Previously it has been demonstrated using models for elastic properties of membranes and lateral pressure profiles of lipid bilayers that the mechanical properties of a lipid bilayer can contribute as much as ∼10 k_BT to the free energy difference associated with a change in protein conformational state. Here, we extend those previous approaches to a more realistic model for a large mechanosensitive channel (MscL). We use molecular dynamics together with the MARTINI model to simulate the open and closed states of MscL embedded in a DOPC bilayer. We introduce a procedure to calculate the mechanical energy change in the channel gating using a three-dimensional pressure distribution inside a membrane, computed from the molecular dynamics simulations. We decompose the mechanical energy to terms associated with area dilation and shape contribution. Our results highlight that the lateral pressure profile of a lipid bilayer together with the shape change in gating can induce a contribution of ∼30 k_BT on the gating energy of MscL. This contribution arises largely from the interfacial tension between hydrophobic and hydrophilic regions in a lipid bilayer.     

Role of hydrophobic forces in bilayer adhesion and fusion.

With the aim of gaining more insight into the forces and molecular mechanisms associated with bilayer adhesion and fusion, the surface forces apparatus (SFA) was used for measuring the forces and deformations of interacting supported lipid bilayers. Concerning adhesion, we find that the adhesion between two bilayers can be progressively increased by up to two orders of magnitude if they are stressed to expose more hydrophobic groups. Concerning fusion, we find that the most important force leading to direct fusion is the hydrophobic attraction acting between the (exposed) hydrophobic interiors of bilayers; however, the occurrence of fusion is not simply related to the strength of the attractive interbilayer forces but also to the internal bilayer stresses (intrabilayer forces). For all the bilayer systems studied, a single basic fusion mechanism was found in which the bilayers do not "overcome" their short-range repulsive steric-hydration forces. Instead, local bilayer deformations allow these repulsive forces to be "bypassed" via a mechanism that is like a first-order phase transition, with a sudden instability occurring at some critical surface separation. Some very slow relaxation processes were observed for fluid bilayers in adhesive contact, suggestive of constrained lipid diffusion within the contact zone.