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There has been much confusion recently about the relative merits of different approaches, osmotic stress, preferential interaction, and crowding, to describe the indirect effect of solutes on macromolecular conformations and reactions. To strengthen all interpretations of measurements and to forestall further unnecessary conceptual or linguistic confusion, we show here how the different perspectives all can be reconciled. Our approach is through the Gibbs-Duhem relation, the universal constraint on the number of ways it is possible to change the temperature, pressure, and chemical potentials of the several components in any thermodynamically defined system. From this general Gibbs-Duhem equation, it is possible to see the equivalence of the different perspectives and even to show the precise identity of the more specialized equations that the different approaches use.

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We used three complementary techniques to vary the chemical potential of water in lipid/water mixtures;we measured the work of removing water from the multilayer lattice formed in water by the zwitterionic phospholipid egg lecithin. By x-ray diffraction, we observed the structural consequences of water removal. There are no discrete classes of "bound water" in this system; the work of removal is a continuous function of water content and lattice repeat spacing. From 30 to 3 A separation between bilayers there exists an exponential "hydration force" repulsion with a 2.6 A decay length. This interaction translates into a very large force to prevent contact between vesicles and planar membranes. It may be an important feature in controlling vesicle-to-cell fusion. As water is removed, bilayers not only move closer, but thicken as the lipid polar groups on the same bilayer move closer together. All cell-cell and vesicle-cell interactions requiring contact between membranes probably involve not only molecular interaction between specific membrane constituents, but also nonspecific interactions. The latter include long-range van der Waals attraction, electrostatic repulsion, and the very strong repulsive force that results from having to remove water from the water-soluble groups that cover and stabilize membrane surfaces (1, 2). Durable contacts, as seen in studies of cell adhesion, must reach beyond or through this "wall" of hydration. For example, sparsely distributed glycoproteins, bearing receptors for specific cell contact, would have to reach through the water coating the cell surface. "Hydration" forces probably stop vesicles from close contact with the cell membrane, making spontaneous fusion difficult (3). The triggering events in exocytosis may be a controlled destruction of vesicle-membrane repulsion which must be more than a simple ionic screening or neutralization of coulombic repulsion (4).We have been exploring all three nonspecific forces acting between phospholipid bilayer membranes. This is done by determining the work needed to remove water from the lamellar lattice formed by phospholipid bilayers in water. In addition to removing water by the osmotic pressure of dextran (1, 2), we now use both a hydrostatic pressure cell and a chamber with controlled vapor pressure to set the chemical potential of water. In using osmotic, hydrostatic, and vapor pressures, we are able to extend our earlier measurements on egg phosphatidylcholine/water multilayers to the full range of water contents.By x-ray diffraction we see that as water is squeezed from the lattice, the bilayers not only come closer together, but deform to get thicker and to bring closer together molecules on the same surface. We show how to divide the work of water removal into separate components for overcoming bilayer repulsion and effecting bilayer deformation. Most of the work goes into pushing bilayers together with progressive water removal, the proportion of the total work going to deform the bilayer diminishes from 16% to 7% as ...

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We have compared hydration forces, electrical dipole potentials, and structural parameters of dispersions of dipalmitoylphosphatidylcholine (DPPC) and dihexadecylphosphatidylcholine (DHPC) to evaluate the influence of fatty acid carbonyl groups on phospholipid bilayers. NMR and x-ray investigations performed over a wide range of water concentrations in the samples show, that in the liquid crystalline lamellar phase, the presence of carbonyl groups is not essential for lipid structure and hydration. Within experimental error, the two lipids have identical repulsive hydration forces between their bilayers. The higher transport rate of the negatively charged tetraphenylboron over the positively charged tetraphenylarsonium indicates that the dipole potential is positive inside the membranes of both lipids. However, the lack of fatty acid carbonyl groups in the ether lipid DHPC decreased the potential by (118 +/- 15) mV. By considering the sign of the potential and the orientation of carbonyl groups and headgroups, we conclude that the first layer of water molecules at the lipid water interface makes a major contribution to the dipole potential.

The behavior of dioleoylphosphatidylethanolamine (DOPE)/cholesterol/tetradecane and dioleoylphosphatidylcholine (DOPC)/cholesterol/tetradecane were examined using x-ray diffraction and the osmotic stress method. DOPE/tetradecane, with or without cholesterol, forms inverted hexagonal (HII) phases in excess water. DOPC/tetradecane forms lamellar phases without cholesterol at lower temperatures. With tetradecane, as little as 5 mol% cholesterol in DOPC induced the formation of HII phases of very large dimension. Increasing levels of cholesterol result in a systematic decrease in the HII lattice dimension for both DOPE and DOPC in excess water. Using osmotic pressure to control hydration, we applied a recent prescription to estimate the intrinsic curvature and bending modulus of the HII monolayers. The radii of the intrinsic curvature, RPO, at a pivotal plane of constant area within the monolayer were determined to be 29.4 A for DOPE/tetradecane at 22 degrees C, decreasing to 27 A at 30 mol% cholesterol. For DOPC/tetradecane at 32 degrees C, RPO decreased from 62.5 A to 40 A as its cholesterol content increased from 30 to 50 mol%. These data yielded an estimate of the intrinsic radius of curvature for pure DOPC of 87.3 A. The bending moduli kc of DOPE/tetradecane and DOPC/tetradecane, each with 30 mol% cholesterol, are 15 and 9 kT, respectively. Tetradecane itself was shown to have little effect on the bending modulus in the cases of DOPE and cholesterol/DOPE. Surprisingly, cholesterol effected only a modest increase in the kc of these monolayers, which is much smaller than estimated from its effect on the area compressibility modulus in bilayers. We discuss possible reasons for this difference.

Amphiphiles respond both to polar and to nonpolar solvents. In this paper X-ray diffraction and osmotic stress have been used to examine the phase behavior, the structural dimensions, and the work of deforming the monolayer-lined aqueous cavities formed by mixtures of dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylcholine (DOPC) as a function of the concentration of two solvents, water and tetradecane (td). In the absence of td, most PE/PC mixtures show only lamellar phases in excess water; all of these become single reverse hexagonal (HII) phases with addition of excess td. The spontaneous radius of curvature R0 of lipid monolayers, as expressed in these HII phases, is allowed by the relief of hydrocarbon chain stress by td; R0 increases with the ratio DOPC/DOPE. Mixtures with very large R0's can have water contents higher than the L alpha phases that form in the absence of td. The drive for hydration is understood in terms of the curvature energy to create large water cavities in addition to direct hydration of the polar groups. Much of the work of removing water to create hexagonal phases of radius R less than R0 goes into changing monolayer curvature rather than dehydrating polar groups. Single HII phases stressed by limited water or td show several responses. (a) The molecular area is compressed at the polar end of the molecule and expanded at the hydrocarbon ends. (b) For circularly symmetrical water cylinders, the degrees of hydrocarbon chain splaying and polar group compression are different for molecules aligned in different directions around the water cylinder. (c) A pivotal position exists along the length of the phospholipid molecule where little area change occurs as the monolayer is bent to increasing curvatures. (d) By defining R0 at the pivotal position, we find that measured energies are well fit by a quadratic bending energy, K0/2 (1/R-1/R0); the fit yields bilayer bending moduli of Kc = (1.2-1.7) X 10(-12) ergs, in good agreement with measurements from bilayer mechanics. (e) For lipid mixtures, enforced deviation of the HII monolayer from R0 is sufficiently powerful to cause demixing of the phospholipids in a way suggesting that the DOPE/DOPC ratio self-adjusts so that its R0 matches the amount of td or water available, i.e., that curvature energy is minimized.