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 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.     

Swelling of phospholipids by monovalent salt

Critical to biological processes such as membrane fusion and secretion, ion-lipid interactions at the membrane-water interface still raise many unanswered questions. Using reconstituted phosphatidylcholine membranes, we confirm here that multilamellar vesicles swell in salt solutions, a direct indication that salt modifies the interactions between neighboring membranes. By varying sample histories, and by comparing with data from ion carrier-containing bilayers, we eliminate the possibility that swelling is an equilibration artifact. Although both attractive and repulsive forces could be modified by salt, we show experimentally that swelling is driven primarily by weakening of the van der Waals attraction. To isolate the effect of salt on van der Waals interactions, we focus on high salt concentrations at which any possible electrostatic interactions are screened. By analysis of X-ray diffraction data, we show that salt does not alter membrane structure or bending rigidity, eliminating the possibility that repulsive fluctuation forces change with salt. By measuring changes in interbilayer separation with applied osmotic stress, we have determined, using the standard paradigm for bilayer interactions, that 1 M concentrations of KBr or KCl decrease the van der Waals strength by 50%. By weakening van der Waals attractions, salt increases energy barriers to membrane contact, possibly affecting cellular communication and biological signaling.     

The interaction and fusion of bilayers formed from unsaturated lipids

The interactions between unsaturated phospholipid bilayers deposited on mica were measured in aqueous solution using a surface forces apparatus. The bilayers were made of L--dioleoylphosphatidylcholine (DOPC), L--dioleoylphosphatidyl ethanolamine (DOPE), and mixtures of the two, and were formed on mica by Langmuir-Blodgett deposition after the lipids were spread on an aqueous substrate from a chloroform solution. The forces are interpreted as electrostatic double-layer and van der Waals forces with long range, and a strong repulsion (hydration or steric force) at distances of several nm. Together they produce a region of weak attraction (a secondary minimum) at 5 nm (DOPE) and 6 nm (DOPC). Fusion of two bilayers into one was observed when the local force per unit area was 2–3 MPa. Other researchers report that phosphatidylethanolamine in vesicles enhances fusion. In this study using deposited bilayers, the presence of DOPE in a DOPC bilayer did not promote fusion, nor did DOPE bilayers fuse more easily than DOPC. The value of the force per unit area at which the two bilayers fuse into one was however decreased by several orders of magnitude when the bilayers were formed from lipids kept in chloroform solution for several days or more. Chromatography showed traces of lipid degradation products in such chloroform solutions.Abbreviations DO dioleoyl- - PE phosphatidyl ethanolamine - PC phosphatidylcholine - FECO fringes of equal chromatic order Of fprint requests to: J. Wolfe     

Measuring electrostatic, van der Waals, and hydration forces in electrolyte solutions with an atomic force microscope

In atomic force microscopy, the tip experiences electrostatic, van der Waals, and hydration forces when imaging in electrolyte solution above a charged surface. To study the electrostatic interaction force vs distance, curves were recorded at different salt concentrations and pH values. This was done with tips bearing surface charges of different sign and magnitude (silicon nitride, Al₂O₃, glass, and diamond) on negatively charged surfaces (mica and glass). In addition to the van der Waals attraction, neutral and negatively charged tips experienced a repulsive force. This repulsive force depended on the salt concentration. It decayed exponentially with distance having a decay length similar to the Debye length. Typical forces were about 0.1 nN strong. With positively charged tips, purely attractive forces were observed. Comparing these results with calculations showed the electrostatic origin of this force.

In the presence of high concentrations (> 3 M) of divalent cations, where the electrostatic force can be completely ignored, another repulsive force was observed with silicon nitride tips on mica. This force decayed roughly exponentially with a decay length of 3 nm and was ~0.07-nN strong. This repulsion is attributed to the hydration force.

     

Direct measurement of the intermolecular forces between counterion-condensed DNA double helices. Evidence for long range attractive hydration forces.

Rather than acting by modifying van der Waals or electrostatic double layer interactions or by directly bridging neighboring molecules, polyvalent ligands bound to DNA double helices appear to act by reconfiguring the water between macromolecular surfaces to create attractive long range hydration forces. We have reached this conclusion by directly measuring the repulsive forces between parallel B-form DNA double helices pushed together from the separations at which they have self organized into hexagonal arrays of parallel rods. For all of the wide variety of "condensing agents" from divalent Mn to polymeric protamines, the resulting intermolecular force varies exponentially with a decay rate of 1.4-1.5 A, exactly one-half that seen previously for hydration repulsion. Such behavior qualitatively contradicts the predictions of all electrostatic double layer and van der Waals force potentials previously suggested. It fits remarkably well with the idea, developed and tested here, that multivalent counterion adsorption reorganizes the water at discrete sites complementary to unadsorbed sites on the apposing surface. The measured strength and range of these attractive forces together with their apparent specificity suggest the presence of a previously unexpected force in molecular organization.     

Measurement and modification of forces between lecithin bilayers.

We probe in two different ways the competing attractive and repulsive forces that create lamellar arrays of the phospholipid lecithin when in equilibrium with pure water. The first probe involves the addition of low molecular weight solutes, glucose and sucrose, to a system where the phospholipid is immersed in a large excess of water. Small solutes can enter the aqueous region between bilayers. Their effect is first to increase and then to decrease the separation between bilayers as sugar concentration increases. We interpret this waxing and waning of the lattice spacing in terms of the successive weakening and strengthening of the attractive van der Waals forces originally responsible for creation of a stable lattice. The second probe is an "osmotic stress method," in which very high molecular weight neutral polymer is added to the pure water phase but is unable to enter the multilayers. The polymer competes for water with the lamellar lattice, and thereby compresses it. From the resulting spacing (determined by X-ray diffraction) and the directly measured osmotic pressure, we find a force vs. distance curve for compressing the lattice (or, equivalently, the free energy of transfer to bulk water of water between bilayers. This method reveals a very strong, exponentially varying "hydration force" with a decay distance of about 2 A.     

Interbilayer interactions between sphingomyelin and sphingomyelin/cholesterol bilayers.

Pressure versus fluid spacing relations have been obtained for sphingomyelin bilayers in the gel phase and equimolar sphingomyelin/cholesterol in the liquid-crystalline phase by the use of X-ray diffraction analysis of osmotically stressed aqueous dispersions and oriented multilayers. For interbilayer separations in the range of 5-20 A, the repulsive hydration pressure decays exponentially with increasing fluid spacing. The decay length (lambda) of this repulsive pressure is about 2 A for both bovine brain and N-tetracosanoylsphingomyelin, similar to that previously found for phosphatidylcholine bilayers. However, both the magnitude of the hydration pressure and the magnitude of the dipole potential (V) measured for monolayers in equilibrium with liposomes are considerably smaller for sphingomyelin than for either gel or liquid-crystalline phosphatidylcholine bilayers. Addition of equimolar cholesterol increases both the magnitude of the hydration pressure and the dipole potential. These data suggest that the magnitude of the hydration pressure depends on the electric field at the interface as given by (V/lambda)2. For sphingomyelin bilayers, there is a sharp upward break in the pressure-fluid spacing relation at an interbilayer spacing of about 5 A, indicating the onset of steric hindrance between the head groups of apposing bilayers.     

Adhesion between cerebroside bilayers

The structure, hydration properties, and adhesion energy of the membrane glycolipid galactosylceramide (GalCer) were studied by osmotic stress/X-ray diffraction analysis.(1) Fully hydrated GalCer gave a repeat period of 67 A, which decreased less than 2 A with application of applied osmotic pressures as large as 1.6 x 10(9) dyn/cm(2). These results, along with the invariance of GalCer structure obtained by a Fourier analysis of the X-ray data, indicated that there was an extremely narrow fluid space (less than the diameter of a single water molecule) between fully hydrated cerebroside bilayers. Electron density profiles showed that the hydrocarbon chains from apposing GalCer monolayers partially interdigitated in the center of the bilayer. To obtain information on the adhesive properties of GalCer bilayers, we incorporated into the bilayer various mole ratios of the negatively charged lipid dipalmitoylphosphatidylglycerol (DPPG) to provide known electrostatic repulsion between the bilayers. Although 17 and 20 mol % DPPG swelled (disjoined) the GalCer bilayers by an amount predictable from electrostatic double-layer theory, 5, 10, 13, and 15 mol % DPPG did not disjoin the bilayers. By calculating the magnitude of the electrostatic pressure necessary to disjoin the bilayers, we estimated the adhesion energy for GalCer bilayers to be about -1.5 erg/cm(2), a much larger value than that previously measured for phosphatidylcholine bilayers. The observed discontinuous disjoining with increased electrostatic pressure and this relatively large value for adhesion energy indicated the presence of an attractive interaction, in addition to van der Waals attraction, between cerebroside bilayers. Possible attractive interactions are hydrogen bond formation and hydrophobic interactions between the galactose headgroups of apposing GalCer bilayers.     

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.     

Interrod forces in aqueous gels of tobacco mosaic virus.

The lateral separation of virus rod particles of tobacco mosaic virus has been studied as a function of externally applied osmotic pressure using an osmotic stress technique. The results have been used to test the assumption that lattice equilibrium in such gels results from a balance between repulsive (electrostatic) and attractive (van der Waals and osmotic) forces. Results have been obtained at different ionic strengths (0.001 to 1.0 M) and pH's (5.0 to 7.2) and compared with calculated curves for electrostatic nad van der Waals pressure. Under all conditions studied, interrod spacing decreased with increasing applied pressure, the spacings being smaller at higher ionic strengths. Only small differences were seen when the pH was changed. At ionic strengths near 0.1 M, agreement between theory and experiment is good, but the theory appears to underestimate electrostatic forces at high ionic strengths and to underestimate attractive forces at large interrod spacings (low ionic strengths). It is concluded that an electrostatic-van der Waals force balance can explain stability in tobacco mosaic virus gels near physiological conditions and can provide a good first approximation elsewhere.     

Modification of anomalous swelling in multilamellar vesicles induced by alkali halide salts

By use of small-angle X-ray scattering it is shown that addition of alkali halide salts in small amounts (0-200 mM) shifts the repeat spacing in multilamellar DC13PC vesicles and alters the anomalous swelling behaviour close to the main transition. Both effects follow the Hofmeister series of the ions. We suggest that the shift of repeat spacing can be explained by ion effects on the van der Waals attractive forces between the membranes and on the decay length of the repulsive hydration force. The anomalous swelling is explained in terms of a critical unbinding of the membranes. The proximity of the critical temperature of the unbinding to the main transition temperature can be tuned by varying the concentration and type of salt in the sample.     

Membrane-Membrane contact: Involvement of interfacial instability in the generation of discrete contacts

The classical approach to understanding the closeness of approach of two membranes has developed from consideration of the net effect of an attractive van der Waals force and a repulsive electrostatic force. The repulsive role of hydration forces and stereorepulsion glycocalyx forces have been recently recognized and an analysis of the effect of crosslinking molecules has been developed. Implicit in these approaches is the idea of an intercellular water layer of uniform thickness which narrows but retains a uniform thickness as the cells move towards an equilibrium separation distance. Most recently an attempt has been made to develop a physical chemical approach to contact which accommodates the widespread occurrence of localized spatially separated point contacts between interacting cells and membranes. It is based on ideas drawn from analysis of the conditions required to destabilize thin liquid films so that thickness fluctuations develop spontaneously and grow as interfacial instabilities to give spatially periodic contact. Examples of plasma membrane behaviour which are consistent with the interfacial instability approach are discussed and experiments involving polycation, polyethylene glycol, dextran and lectin adhesion and agglutination of erythrocytes are reviewed.     

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.     

Differences between non-specific and bio-specific, and between equilibrium and non-equilibrium, interactions in biological systems

The interaction forces between biological molecules and surfaces are much more complex than those between non-biological molecules or surfaces, such as colloidal particle surfaces. This complexity is due to a number of factors: (i) the simultaneous involvement of many different molecules and different non-covalent forces - van der Waals, electrostatic, solvation (hydration, hydrophobic), steric, entropic and 'specific', and (ii) the flexibility of biological macromolecules and fluidity of membranes. Biological interactions are better thought of as 'processes' that evolve in space and time and, under physiological conditions, involve a continuous input of energy. Such systems are, therefore, not at thermodynamic equilibrium, or even tending towards equilibrium. Recent surface forces apparatus (SFA) and atomic force microscopy (AFM) measurements on supported model membrane systems (protein-containing lipid bilayers) illustrate these effects. It is suggested that the major theoretical challenge is to establish manageable theories or models that can describe the spatial and time evolution of systems consisting of different molecules subject to certain starting conditions or energy inputs.     

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.     

A statistical mechanical treatment of fatty acyl chain order in phospholipid bilayers and correlation with experimental data. A. Theory

A theoretical model has been developed in order to describe the organization of acyl chains in phospholipid bilayers. Since the model is intended to reproduce highly quantitative experimental results such as the deuterium magnetic resonance (NMR) data and to supplement the experimental information, all the rotameric degrees of freedom, the excluded volume interactions and the van der Waals interactions have been considered. The model is a direct extension of a generalized van der Waals theory of nematic liquid crystals to flexible molecules. In this picture, the anisotropy of the short-range repulsive forces which are treated by a hard core potential is introduced as the dominant factor governing intrinsic order among the chains. The anisotropy of the attractive forces, which are approximated by a molecular field, plays a somewhat secondary role. The dependence of the energy of interaction on the relative chain conformations is approximated by two order parameters reflecting respectively the ‘average shape’ of the molecules and the ‘average shape’ in a ‘mean orientation’. The influence of the interactions in the polar region on the lateral chain area is accounted for by an effective lateral pressure. In certain aspects the model has features in common with the Mar?elja theory.     

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.     

Repulsive stabilization in black lipid membranes. A hydrodynamic model

A linear stability analysis is performed for a black lipid membrane. The hydrodynamic model consists of a viscous hydrocarbon film sandwiched between two aqueous phases. Attractive forces (van der Waals and electrical) and repulsive forces (steric) are expressed as body forces in the equations of fluid motion in the three phases. The steric repulsion due to overlap of the hydrocarbon chains of the lipids at small film thicknesses is described via an exponentially decaying interaction potential. The dispersion equation displays two modes of vibrations: the bending mode with the two Film surfaces transversely in phase, and the squeezing mode with the two surfaces 180 degrees out of phase. For symmetrical films, these two modes are uncoupled, and the squeezing mode (with thickness variations) is stabilized by the repulsive interactions. For nonsymmetrical films (different surface tensions, surface charges, etc.). these two modes are coupled and the asymmetry induces a shift of the marginal stability curve to shorter wavelengths.     

Protein separation by potential barrier chromatography

Potential barrier chromatography (PBC) is a liquid chromatographic method in which the adsorption and desorption of proteins are controlled by modifications of repulsive double-layer and attractive van der Waals interactions between the proteins and the adsorbent through changes in mobile phase composition. In this review PBC is compared and contrasted to the more traditional chromatographic methods, namely, ion exchange, gel permeation, hydrophobic interaction, and affinity chromatography. The physical forces that underlie PBC (as well as the other methods) are discussed in terms of their effects on chromatographic behavior. Experimental results are presented to illustrate the use and simplicity of the method.