Polymorphism of Lipid-Water Systems

CHAPTERS

Polymorphism of Lipid-Water Systems J.M. SEDDON and R.H. TEMPLER Department of Chemistry, Imperial College, Exhibition Road, London SW7 2AY, U.K.

©1995 Elsevier Science B. V All rights reserved

Handbook of Biological Physics Volume I, edited by R. Lipowsky and E. Sackmann 97

Contents 1. Introduction

99

2. Interfacial curvature

101

3. Structure of lyotropic phases

105

3.1.

Phase identification

105

3.2. 3.3. 3.4. 3.5.

Topology determination Phase dimensions Nomenclature for phase structures Crystalline phases

107 110 110 110

3.6. 3.7.

Ordered lamellar phases Fluid phases

112 115

3.8. Isotropic solution phases 4. Phase behaviour

120 123

4.1.

Lyotropic phase diagrams

4.2.

Phase stability

123 126

4.3. Packing geomeUy and frustration 4.4. Curvature elastic energy 4.5. Lateral stress profile 4.6. Defects and epitaxiality in phase transitions 5. Factors affecting lyotropic transitions

126 129 130 134 136

5.1.

Types of transition

136

5.2. 5.3. 5.4. 5.5. 5.6.

Effect of lipid chemical structure Lipid mixtures Solution effects Solute effects Phase metastability

137 138 139 139 141

5.7.

Transition kinetics

141

6. Biological implications 6.1. Non-lamellar phases in biology 6.2. Membrane fusion and cell signal transduction 6.3. Homeostatic control of *phase stability* 6.4. Bilayer stress profile and regulation of membrane protein activity 6.5. Protein/lipid mixtures 7. Open problems Acknowledgements References

98

143 143 145 145 146 146 146 149 149

1. Introduction This chapter will describe the types of liquid-crystalline phases adopted by lipids in water, and the factors which control phase stability. Even single lipid systems can display a quite extraordinarily rich variety of liquid-crystalline phase structures upon varying the water content and/or the temperature. These different phases result from an optimization of the hydrophobic effect with a variety of intra- and intermolecular interactions, in combination with a number of geometric packing constraints. Examples of lyotropic phase structures are the fluid lamellar La phase, fig. la, the inverse hexagonal Hn phase, fig. lb, and the inverse bicontinuous cubic phase of crystallographic spacegroup Pn3m, fig. Ic. In this chapter we will deal primarily with the fluid lyotropic phases, since these are likely to be of the most direct relevance to the structure and function of biomembranes. The properties of lamellar phases (those based on lipid bilayers) are discussed extensively in various other chapters in this volume; therefore, in this chapter the emphasis will rather be on the various non-lamellar phases, whose roles in biomembrane structure and function are still controversial and poorly understood. Furthermore we will focus attention on biological lipids such as phospholipids, rather than dealing with all surfactant systems. However, much of the behaviour described here is of quite general relevance to lyotropic systems: most if not all of the structures formed by biological lipids can also be observed in simpler surfactants, under appropriate conditions.

•.

*

'f""-" > ^ -

Fig. la. Examples of lyotropic structures: La fluid lamellar phase. 99

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JM Seddon and R.H. Templer

Fig. lb. Examples of lyotropic structures: inverse hexagonal Hn phase (from [1]).

Fig. Ic. Examples of lyotropic structures: inverse bicontinuous cubic phase Pn3m (from [2]).

Polymorphism of lipid-water systems

101

Although biopolymers such as DNA also form lyotropic liquid crystalline phases, the mechanisms are quite distinct from those applying to amphiphiles, and such systems will not be described here. Much of the experimental work on lipid systems has been performed on pure, well-defined synthetic lipids, although natural lipid extracts from membranes often exhibit the same phase structures, notwithstanding the fact that they usually consist of very complex mixtures of different lipids. The structures of the translationally ordered lipid phases have been reviewed a number of times [1-10], and these articles should be consulted for further details. A recent issue of Chemistry and Physics of Lipids [11] was devoted to the subject of lipid polymorphism, and an issue of Journal de Physique [12] to geometry and interfaces, relating mainly to lyotropic liquid crystals. Books have appeared on the subject of phospholipid bilayers [13] and the physics of amphiphilic layers [14, 15], and theoretical approaches to membrane conformations have been recently reviewed [16, 17]. 2. Interfacial curvature In order to describe and characterize the various lyotropic phases, it is most useful to focus attention on the interface between the polar and non-polar regions of the phases (i.e. the narrow region where the headgroups are attached to the hydrocarbon chains), corresponding to the plane at which the interfacial tension acts within a monolayer. (By interfacial tension we mean purely the tension at the hydrocarbon/water interface. However, many authors use this term to mean the total net lateral tension, i.e. they include the lateral stress contributions from chain-chain and headgroup-headgroup lateral repulsions and/or attractions.) This interfacial plane should lie close to the neutral surface, i.e. the surface at which there is no change in area per molecule upon bending. Apart from its area, the interface is characterised by its mean and Gaussian curvatures, H and K. These are related to the principal curvatures c\ and C2 at a given point P on the surface, fig. 2, by H = [ci + C2]/2,

(1)

i ^ = CiC2.

(2)

Different phases have different values of mean and/or Gaussian interfacial curvatures, and these may or may not be uniform at different points on the interface within a single phase. We will adopt the convention that for a Hpid monolayer, if > 0 denotes curvature towards the chain region, whereas ff < 0 denotes curvature towards the water region; see fig. 2. Note that although this definition for a monolayer is unambiguous, the sign of the mean curvature for a lipid bilayer is arbitrary. The mean curvature H of a monolayer can be changed simply by bending, without stretching the interface. However, changing the Gaussian curvature K necessarily involves stretching (or contracting) the interface (but this could be achieved at constant interfacial area if the molecules are free to redistribute laterally). Both of these types of deformation involve associated curvature elastic energy costs.

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J.M. Seddon and R.H. Templet

C,, Cg > 0

H > 0 (K > 0 )

(K > 0)

Fig. 2. Definition of mean curvature H and Gaussian curvature K for a lipid monolayer. Ri and Ri, and c\ and C2, are the principal radii of curvature, and the principal curvatures, respectively, at the point P. n is the unit normal vector of the surface patch A at point P , directed in the positive z-direction. Adapted from [1].

103

Polymorphism of lipid-water systems

C-, < 0 C2 > O

K < 0 (H

or = 0 )

Apex^"

Saddle point

Fig. 3. Saddle surface, of (non-uniform) negative Gaussian curvature. From [1].

The Gaussian curvature Jf is a more fundamental property of the interface than H since it determines the qualitative nature of the surface. Surfaces for which K is positive are known as elliptic, and naturally bend round to form closed shells. A micelle or an inverse micelle are examples of this. When either of the principal curvatures are zero, the Gaussian curvature is zero, and the surface is known as parabolic. The lamellar and hexagonal phases are examples of this. The third possibility arises when the principal curvatures c\ and C2 are of opposite sign, leading to a negative Gaussian curvature. These surfaces are known as hyperbolic, and an example is the saddle surface, shown in fig. 3. The Gaussian curvature is most negative at the saddle point

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J.M. Seddon and R.H. Tempter

Schwarz's P-surface (Im3m)

Fig. 4. An example of an infinite periodic minimal surface: Schwartz's P-surface.

and increases smoothly to zero at the four apices. When the principal curvatures are everywhere equal in magnitude but opposite in sign, then the surface has zero mean curvature at all points and is known as a minimal surface. Such surfaces can be extended to fill space, forming infinite periodic minimal surfaces, which form a single septum, dividing space into two congruent sub-volumes. An example is the Schwarz P-surface, shown in fig. 4. Thus when K
Polymorphism of lipid-water systems

105

Fig. 5. A bilaycr draped on a saddle surface has a smaller area at the centre of each monolayer hydrocarbon chain region (ari) than at the bilayer mid-plane (S), and an even smaller area {ai) at each headgroup region. From [31].

3. Structure of lyotropic phases 3,1. Phase identification Diffraction methods, in particular X-ray scattering, are the most reliable way of carrying out lipid phase identification. Spectroscopic techniques such as NMR have been used by certain authors for phase identification, although this can under certain circumstances lead to incorrect assignments. Freeze-fracture electron microscopy, when used in conjunction with X-ray diffraction, can yield useful complementary data [32]. In the characterization of lipid mesophases by diffraction, there are two regions of the diffraction pattern that are used to identify the phase. The small angle region identifies the symmetry and long range organization of the phase, whereas the wide angle region gives information on the molecular packing, or short range organization of the phase. The signature of a translationally ordered mesophase is the appearance of one or more sharp (Bragg) peaks in the low-angle region of the diffraction pattern.

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J.M. Seddon and R.H. Templer

The long-range translational ordering of the lipid/water aggregates (bilayers, cylinders, micelles, etc) onto 1-, 2- or 3-dimensional lattices gives rise to Bragg reflections whose reciprocal spacings (shki = 1/dhki) are in characteristic ratios; see fig. 1, for example: Lamellar: si = l/d (Ratios 1, 2, 3, 4,...) Hexagonal: Shk = 2{h} + A:^ - hkf^^/y/3a (1, Vs, 2, \/7, 3, \/T2, \fvi,...) Cubic: sm^{h^^k^^i^y^^la (1, V2, v^, 2, \/5, V^, \/8, 3,...) Once the lattice type has been identified, it is necessary to determine the crystallographic spacegroup [33] to which the phase belongs, from the pattern of systematic absences in the diffraction pattern. However, this is often not trivial, since usually only a few low-angle Bragg peaks are detected, due to the large thermal disorder inherent in liquid-crystalline phases, which strongly damps the intensities at larger diffraction angles. In fact, there are a number of examples in the literature where incorrect phase assignments appear to have been made. For the 2D phases (hexagonal, rectangular, square, oblique) there are only 17 possible plane groups. For 3D phases there are 230 possible space groups, although very few of these have so far been observed in lyotropic systems. To date, six different cubic phases have been clearly identified in lipid systems, belonging to different spacegroups (it should be noted that totally different phase structures could in principle have the same spacegroup, although for lipids this usually seems not to occur). A number of other cubic phases have been tentatively identified in various systems. From unaligned samples it is usually only possible to identify the cubic aspect from the systematic absences, leaving an ambiguity about the precise spacegroup. In some cases this ambiguity could be resolved if monodomain samples were available, since hkl reflections are not fully permutable for certain spacegroups (i.e. the observed intensity (hkl) may not be equal to that of (khl), the non-cyclic permutation). Furthermore, complementary freeze-fracture electron microscopy experiments can be of help, by showing directly the presence of mirror planes in the phase structure [29, 32]. The intensities of the various Bragg peaks are determined by the distribution of matter (electron density) in the unit cell, which is constrained by the symmetry of the spacegroup. A symmetry-allowed reflection may nonetheless have zero intensity because the unit cell Fourier transform happens to pass through zero at that particular diffraction angle. If it is possible to deduce the phasing of the structure factors (from the intensities of the Bragg peaks) then low-resolution (electron) density maps can be directly obtained by Fourier transformation. However, the standard direct methods, or isomorphous replacement, for phasing are not generally suitable for liquid crystalline systems. In some cases, in particular for lamellar phases, it is possible to use water swelling experiments to deduce the phasing [34]; in neutron diffraction experiments, the solvent contrast variation technique may be employed [35]. However, for 3D structures such as lyotropic cubic phases, the only successful technique so far for phasing the reflections has been to employ a pattern recognition approach [29, 36]. A further major problem with the characterisation of lipid phases is the difficulty of ensuring that the sample is at equilibrium. In part this may be due to the rate at which

Polymorphism of lipid-water systems

^07

a phase comes to equilibrium being very slow. However, a further problem is that the phase itself may be metastable, reverting to more stable forms over a time scale which can span seconds to months. For example, both the gel and fluid lamellar phases of phosphatidylethanolamines are metastable within certain temperature ranges, and will spontaneously convert to lamellar crystals on incubation [37]. 3.2. Topology determination There are three main aspects to the determination of the topology of a lipid mesophase (we use the term topology here in a very loose sense). 3.2.1. Sign of the interfacial mean curvature Firstly, it is vital to establish whether the phase is inverse or normal, i.e. whether the polar/non-polar interface curves on average towards the water or towards the hydrocarbon region of the phase. With the exception of the lamellar L^ phase, all of the lyotropic liquid-crystalline phases, for example the hexagonal phases, fig. 6, may potentially occur either as type I (normal, oil-in-water) structures, or as type II (inverse, water-in-oil). Surprisingly, because of Babinet's principle, it is not trivial to determine by diffraction which type one is dealing with. However, there are a number of approaches which can be used in order to establish it with reasonable certainty [1,3]: a. If a fluid non-lamellar phase is observed to coexist with an excess aqueous phase, this is by itself strong evidence for an inverse, type II structure; type I phases of lipid systems almost invariably break up into micellar solutions beyond a certain limiting water content. b. If a fluid non-lamellar phase occurs to lower (higher) water content than the lamellar L^ phase, then it is probably inverse (normal). This rule is however dangerous, as there are important exceptions to it. For example, the phase diagram of monoolein exhibits two inverse bicontinuous cubic phases on the high water side of the L^ phase. c. If a phase of unknown type (normal or inverse) is adjacent in the phase diagram to a non-lamellar phase of known type, then it almost certainly is the same type. d. If the interfacial area per molecule (deduced from diffraction data) is plotted against water concentration, it should not decrease with increasing water content, if the assumed type is correct. e. The value of interfacial area per molecule at the water/lipid interface is normally lower (higher) for an inverse (normal) phase than for an adjacent La phase, either when the transition is driven by varying the composition, or by varying the temperature. f. The variation of the intensities of the Bragg peaks for a range of water contents can provide unambiguous evidence for whether the phase is normal or inverse. g. It should in principle be possible to probe phase type by employing neutron diffraction contrast variation techniques, although as yet this has been little employed in structural studies of lipid polymorphism.

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a)H,

o2o »

Fig. 6. Non-lamellar phases may exist as nonnal (oil-in-water) or inverse (water-in-oil) types, (a) An example of the former is the hexagonal phase Hj. From [1]. (b) An example of the latter is the hexagonal phase H]]. For this phase, water swelling leads to a strong increase in interfacial area per molecule. From [1].

3,2,2. Genus Secondly, for complex 3D structures such as cubic phases, it is necessary to establish whether the phase is bicontinuous or discontinuous (micellar). These two possibilities are topologically distinct: the former type has a single 'surface', but many holes, the latter has n^ separate surfaces (per unit cell), each free from holes in the interface. Furthermore, bicontinuous phases may have different values of genus. The genus p of a phase is a fundamental topological property related to the connectivity of the surface defining the lipid layers (e.g., a minimal surface for bicontinuous cubic phases). It is defined as the maximum number of non-intersecting closed loop cuts that can be made in the surface without it falling into two parts. For an in-

Polymorphism of lipid-water systems

109

finite periodic minimal surface, the genus is, strictly speaking, infinite. However, the genus per unit cell is well defined, and is determined by using the translational symmetry to connect together corresponding surfaces cut by opposite faces of the primitive translation cell of the surface. Thus the number of topologically distinct holes per unit cell gives the genus g of the phase. For the P, F, and gyroid minimal surfaces, the genus per unit cell is 3 [38]; in fact they have to have the same value because they are connected by the Bonnet transformation. It should be noted that the space group and the primitive translation cell of the underlying minimal surface need not be the same as that of the cubic phase itself. For example the F-surface underlying the Pn3m (= Q'^'^^) cubic phase has space group F43m (= Q^^^) and has a primitive translation cell with twice the volume of the Pn3m cubic phase. A most useful quantity is the dimensionless area, Ao, per conventional unit cell of the cubic phase, which has the values 1.919, 2.345 and 3.091 for the F, P and gyroid minimal surfaces, respectively. Thus the total area per unit cell at the bilayer centre is given by a^-Ao, where a is the conventional unit cell lattice parameter, and the area per molecule at this interface follows directly if the water content of the phase is known. The Gauss-Bonnet theorem relates the number of disconnected surfaces ns, each having the same genus g, to the surface integral of the Gaussian curvature by: / /

KdA^Am,{\^g).

(3)

Thus the bicontinuous phases have a negative average Gaussian curvature, whereas for the micellar cubic phases this quantity is positive. Within the family of bicontinuous phases, certain spacegroups could correspond to a variety of different structures, having different degrees of connectivity (genus) [30, 39]. For example, the cubic phase Im3m (= Q^^^) could correspond to structures based on surfaces of genus per unit cell of 3 (P-surface), 4 (I-WP-surface), 9 (Neovius surface) or 10 (0,C-TO-surface). In principle it should be possible to distinguish these from the relative intensities of the observed Bragg peaks, although the form factors have so far only been calculated for the first of these surfaces [40]. The diffraction patterns from the bicontinuous cubic phases based on the P-, F- and G-surfaces tend to have quite characteristic intensity distributions [29], and when an unusual intensity distribution is observed, the possibility that a higher genus phase is present should be considered. Further evidence may be obtained from consideration of whether the dimensions of the phase are compatible with the composition and the packing properties of the amphiphile (e.g., maximum molecular length). Such analysis suggests that both genus 3 [41] and higher genus cubic phases [42] occur in certain ternary surfactant/oil/water systems. Even in binary systems such as sodium dodecyl sulphate/water, the structure of the Im3m cubic phase reported [43] is uncertain, but appears not to be based on the genus 3 P-surface [30]. 3.2,3. Monolayer or bilayer structure Thirdly, for the bicontinuous phases the structure could in principle be based either on a lipid bilayer, or on a monolayer [30]. Note that for the hexagonal and micellar cubic phases the structure is normally based on a lipid monolayer. For most biological

110

J.M. Seddon and R.H. Tempter

lipid systems (at least in the absence of added oil or co-surfactant) the bicontinuous structures appear to be based on bilayers, whereas for some surfactant or microemulsion systems, the picture is much less clear, and both types of bicontinuous phase might occur [30]. For example, in the ternary system didodecyldimethylammonium bromide/octane/water, there is some evidence for a monolayer cubic phase structure based on an underlying I-WP minimal surface (of genus 4) [44]. 5.3. Phase dimensions Although a detailed analysis of the diffraction intensities is required to obtain the mesophase structure in terms of electron density maps, useful structural information such as the lipid and water layer thicknesses, and the interfacial area per molecule, can be obtained simply from the positions of the diffraction peaks [3]. The thicknesses are ill-defined to the extent that the water-lipid interface is not completely sharp. For non-lamellar phases the value of area per molecule depends on the position chosen for the interface. Often this is a hypothetical, sharp interface dividing the lipid layer from the water. If this becomes unreaHstic, for example when the lipid headgroups are very elongated, it may be more appropriate to lump the polar headgroups together with the water, setting the interface at the polar/non-polar boundary. Such analysis requires knowledge of the composition of the phase and the densities of the water and lipid components. The density of the water is usually assumed to be the same at a given temperature as that of bulk water, while the density of the lipid may be measured either by the oscillating tube or neutral bouyancy techniques. For more complex structures such as the bicontinuous cubic phases, the various dimensions may be estimated using results from differential geometry [30]. 3.4. Nomenclature for phase structures The most widely used nomenclature for lyotropic phases is that proposed by Luzzati [3], and this will be adopted here. The lattice type is denoted by a capital letter, e.g., L for lamellar, H for hexagonal and Q for cubic. Subscripts I and 11 are used to denote normal (oil in water) or reversed (water in oil) topology phases. A Greek subscript is used to denote the chain conformation: c for crystalline, /? for ordered gel-like, a for liquid-like, a/3 for coexisting gel- and liquid-like regions, and 6 for a helically coiled chain conformation. A list of the well-established lyotropic phase types is given in table 1. There is actually a family of cubic phases, and those discovered to date, whose structures are well-established, are listed in table 2. 3.5. Crystalline phases Most phospholipids form crystalline lamellar Lc phases at low temperatures and/or hydrations. These phases exhibit both long- and short-range order in three dimensions and are therefore true crystals. They may be anhydrous, or may also contain a number of water molecules of co-crystallization. Many lipid structures have now been solved by single crystal studies [45].

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Polymorphism of lipid-water systems

Table 1 List of the well-established, translationally ordered lyotropic phases. Reference

Phase

Description

Lc l^ L0 L^/

3D lamellar crystals Lamellar stack of 2D crystalline bilayers Lamellar gel (untitled) Lamellar gel (tilted) Interdigitated gel Partial gel Lamellar phase of square-packed, helically-coiled (S) chains Rippled gel phase Ribbon phase with ^-packed chains Fluid lamellar phase Hexagonal Hexagonal, complex Rectangular Oblique Cubic Tetragonal Rhombohedral

L/SI La/9

U P^' P6 La

H H^ R M Q T Rh

45 46 47 47 48 3 47, 49 47 47 3 1 3 43 43 29 3 3

Table 2 List of the well-established cubic lyotropic phases. Spacegroup symbol P4332 Pm3n Pn3m Fd3m Im3m Ia3d

Spacegroup number

Cubic aspect

Reference

0^12

3 5 4 15 8 12

29 50 29 36 29 29

Q223 Q224 Q227

0229 Q230

The molecular conformation in the lamellar crystalline phases of dilauroylphosphatidylethanolamine (DLPE) [51], and dimyristoylphosphatidylcholine (DMPC) [52] are compared in fig. 7. Although the conformations appear quite similar, methylation of the phospholipid terminal ammonium group does in fact have a profound effect on the molecular packing and interactions. It is striking that the intermediate methylated compound, dilauroylphosphatidyl-Ar,iV-dimethylethanolamine (dimethyl-DLPE) [53] has a quite different conformation, as shown in fig. 8, with the headgroups being interdigitated. All three lipids pack into bilayers, but for DLPE a tight network of headgroup-headgroup hydrogen-bonds is formed, fig. 9, with the headgroups parallel to the plane of the layer, whereas DMPC headgroups (lacking any donor groups) interact via bridging water molecules. Certain lipids when incubated in water at low temperatures, adopt so-called subgel phases. These appear to consist of lamellar stacks of two-dimensional crystalline

112

JM. Seddon and R.H. Templer

DLPE

DMPC

Fig. 7. Molecular conformation in the lamellar crystal phase of DMPC. From [45].

bilayers. For a charged lipid such as phosphatidylglycerol (PG), the crystalline bilayers can be swollen apart in water by electrostatic repulsion [46]. 3,6. Ordered lamellar phases Many lipids adopt lamellar phases at low temperatures in which the hydrocarbon chains are still ordered essentially in the dW-trcms conformation, but where they undergo hindered long-axis rotation on a time scale of 100 nsec. The effective cylindrical symmetry means that the chains pack onto 2D hexagonal lattices. The headgroups are normally disordered and the lateral correlations between adjacent layers are weak or nonexistent. Such gel phases are normally formed in the presence of water, although this is not always strictly necessary. The thickness of the water layer depends on factors such as the water content and temperature, and the lipid headgroup size, polarity and charge. The maximum water content is often relatively low, with a water layer thickness in the region of 8-16 A, although the gel phases of charged hpids can swell to very large layer spacings. In the L^ gel phase, fig. 10a, the hydrocarbon chains are arranged parallel to the layer normal, with a value close to 20 A^ for the cross-sectional area per chain. The hp> phase, fig. 10b, is a tilted version of L/j, and different tilt directions with respect to the underlying hexagonal lattice may occur. The tilting occurs when the headgroup area packing requirement exceeds twice that of the chains (for diacyl lipids): tilting allows the packing mismatch to be accommodated. However, when the tilting becomes too great, then the (untitled) interdigitated L/31 phase, fig. 10c, may

Polymorphism of lipid-water systems

113

Fig. 8. Structure of the headgroup-interdigitated lamellar crystal phase of dimethyl-DLPE. The positions of the L- and D-enantiomers are indicated. From [53].

form, which has a similar cross-sectional area per chain to Lp, but with approximately twice the area available per headgroup. A gel phase where the lamellae are deformed by a periodic modulation is not uncommon. This P^/ ripple phase, fig. lOd, occurs below the L^ phase with temperature. It has been observed in phosphatidylcholines [47] and phosphatidylglycerol at neutral pH [56], and in phosphatidylethanolamine [57] and phosphatidic acid [58] at

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JM. Seddon and R.H. Tempter

DLPE

t I

7.8 A

-asAFig. 9^ Headgroup packing and hydrogen bonding in crystalline DLPE. The molecular area S is 38.6 A^. The b and c unit cell parameters are indicated, along with a number of intramolecular and intermolecular contact distances. From [54].

f?f?f?(?(?f?f?f?P

Lp SSSdSSSSS

f?f?f?f?f?l?f?f?l? Lpi SSSSSSSSa Fig. 10. Gel phases of lipids: a) L^ untitled gel; b) L^/ tilted gel; c) L^j interdigitated gel; d) P^/ rippled gel. Adaptedfrom[55].

Polymorphism of lipid-water systems

115

high pH. The lattice is usually oblique (2D space group p2, No. 2), with the chains essentially being in the tilted gel-like /3' conformation. An unusual lamellar he phase has been observed in dry phosphatidylchohnes [47, 49]. Here the hydrocarbon chains are coiled into hehces and are arranged on a two-dimensional square lattice. The polar headgroups are also arranged on a square lattice (the length of which is the diagonal of the square lattice of chains), and are oriented perpendicular to the layer, interdigitated with those from the apposed neighbouring bilayer. A closely related phase denoted P^ (2D space group cmm. No. 9) is also found in dry phosphatidylcholines. In this phase, the hydrocarbon chains have the 6 conformation, and ribbon-like strips of bilayer are packed onto a 2D centred rectangular lattice. J. 7. Fluid phases Upon heating a gel phase lipid, the cross-sectional area per chain characteristically increases to a limiting value of about 21 A^; upon further heating, the chains melt to a liquid-like conformation, transforming usually to the lamellar L^ phase, fig. 11

ill water

oil

wimm Fig. 11. The fluid lamellar La phase and its water- and oil-swollen versions.

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JM Seddon and R.H. Templer

(For some lipid systems the gel phase melts directly to a fluid non-lamellar phase, for example Hn or cubic). The interfacial area per molecule expands by 15-30% on transforming to the LQ phase (the expansion is usually smaller for the Hn phase, and intermediate for the inverse bicontinuous cubics), and there is the onset of rapid lateral diffusion (Dtrans « 10""^^ m^sec"^). The La phase may be swollen by addition of water or oil up to certain limiting spacings. For uncharged phospholipids, the maximum water layer thickness is typically in the range of 10-30 A. However, certain lyotropic lamellar phases will swell to extremely large spacings (as large as 5000 A) upon addition of oil or water [59]. The swelling may be driven electrostatically (for charged bilayers), but can also result from thermally excited undulations if the layers are quite flexible. It should be noted that oil-swelling separates the bilayer into two monolayers. 5.7.7. 2Dfluidphases If thefluidlipid aggregates consist of indefinitely long cylinders (not necessarily of circular cross-section) rather than bilayers, then two-dimensional fluid phases will be formed. The simplest and best established of these are the normal and inverse hexagonal phases Hi and Hn (2D space group p6m. No. 17) shown in fig. 6. In the HI phase the lipids aggregate into circular cylindrical micelles which pack onto the hexagonal lattice, with a continuous water region filling the volume between the cylinders. In the inverse Hn phase on the other hand, the cyhnders contain water cores surrounded by the lipid polar headgroups, with the remaining volume completely filled by the fluid hydrocarbon chains at an essentially uniform liquid alkane density. Although the Hi phase is very common in simple surfactant systems, it tends not to be formed by diacyl phospholipids, although it is observed within certain hydration ranges in lyso-phospholipids. The Hii phase is very common in phospholipids such as PE, having small weakly hydrated headgroups, and having attractive headgroup-headgroup interactions [1]. It is also observed in hydrated phospholipid/amphiphile systems such as PC/fatty acid mixtures [60-64]. Although the vast majority of reported hexagonal phases are based on aggregates having a single curved lipid layer (monolayer), a more complex type, denoted H^, has been found in certain systems, whose structure appears to be based on a hexagonal packing of cylinders formed by curved lipid bilayers [3]. For some systems, the shape of the cylinders may deviate from circular in crosssection, leading to a packing into 2D phases of lower symmetry, such as rectangular or oblique [43]. 3 J. 2. 3Dfluidphases The vast majority of three-dimensional fluid phases so far detected are of cubic symmetry, although rhombohedral, tetragonal and orthorhombic phases of inverse topology have been detected in a few lipid systems at low hydrations [3]. In the rhombohedral phase (space group R3m, No. 166). short segments of lipid/water cylinders are connected three by three to form planar hexagonal networks, which stack into a trilayer structure, fig. 12a. The tetragonal phase (space group 1422, No. 97) is similar, but the cylinders are connected four by four to form planar square

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Polymorphism of lipid-water systems

RH (R3m)

Fig. 12a.

Three-dimensional non-cubic liquid-crystalline phases: rhombohedral (spacegroup R3m). From [1].

networks, which stack into a body centred tetragonal lattice, fig. 12b. A bodycentred orthorhombic phase (space groups mmm, No. 47 or 222, No. 16) has been observed in certain anhydrous soap systems [3], but has not as yet been found in phosphoHpids. By far the largest family of 3D lyotropic phases are of cubic symmetry. These phases may be easily detected by polarising microscopy, since they are optically isotropic, and are very viscous, unlike isotropic or micellar solutions. To date five centrosymmetric cubic phases have been well characterised in lipid/water systems, along with a further non-centrosymmetric one which occurs in certain lipid/protein mixtures at low hydration. These phases are listed in table 2, along with their spacegroup number and cubic aspect. Strictly speaking it is only possible to deduce the cubic aspect from the pattern of systematic absences in the powder-like diffraction patterns, but it is assumed that the most symmetric spacegroup within a given cubic aspect is the correct one [29].

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J.M. Seddon and R.H. Templer

T, (1422)

Fig. 12b.

Three-dimensional non-cubic liquid-crystalline phases: tetragonal (spacegroup 1422). From [1].

There appear to be two distinct families of cubic phases. One type is bicontinuous, and is based on underlying periodic minimal surfaces; the other type is micellar, being based on complex packings of discrete micellar aggregates. Both types may be normal (oil-in-water) or inverse (water-in-oil), although curiously, apart from Ia3d ( = Q^^^), a given spacegroup usually exists as only one type or the other. There are many other examples of cubic phases that are not yet definitely identified, and it is probable that more cubic phases remain to be discovered. In terms of minimal surfaces, the inverse bicontinuous cubic phases Ia3d, Pn3m and ImSm are formed by draping a continuous lipid bilayer onto the gyroid, F- and P-minimal surfaces, respectively. It is interesting that these three surfaces constitute a family of infinite periodic minimal surfaces which are related to each other by the Bonnet transformation. This means that one surface can be transformed into either of the others simply by bending, which leaves the Gaussian curvature at all points unchanged, and preserves all angles, distances and areas on the surface [18]. However, in our opinion it is unlikely that cubic-cubic transitions occur by a Bonnetlike transformation, which would require unphysical layer self-intersections to occur.

Polymorphism of lipid-water systems

119

The lattice parameters observed so far for cubic phases fall into the range of 8 0 270 A, both for ternary surfactant/oil/water systems [42] and for lipid mixtures [65]. There are some theoretical grounds for believing that the latter figure may be close to an upper stability limit [66], but this remains to be established. Ia3d (= Q^^^) was the first cubic phase structure to be solved [67]. The inverse type, shown in fig. 13a, is formed by a number of lipid systems, whereas the type I (oil-in-water) version is rather common in surfactant systems. The structure consists of two interwoven yet unconnected chiral networks of water/lipid cylinders, connected coplanarly three by three and separated by the G-minimal surface. Although the two networks are chiral, the cubic phase itself is centrosymmetric. The structure of Pn3m (= Q*^^^) was determined independently by two groups [22, 68]. It consists of two interwoven tetrahedral networks of water channels arranged on a double-diamond lattice, separated by the F-minimal surface; see fig. 13b. The third bicontinuous cubic phase, Im3m (= Q^^^) has orthogonal networks of water channels connected six-by-six, and separated by die P-minimal surface, fig. 13c. Pn3m seems always to be inverse, and Im3m is usually so, although one example of a type I (oil-in-water) Im3m cubic phase has been reported [43]. The cubic phase Pm3n (= Q^^^) occurs in certain systems adjacent to the micellar solution, and its structure has been controversial since the original proposal [69] shown in fig. 14a. It is now agreed that the structure in fact consists of a cubic packing of two types of micelle [70]. There are 2 quasi-spherical, and 6 slightly asymmetric micelles per unit cell. However, it is not yet fully established whether the asymmetric micelles are disk-like, as shown in fig. 14b [50, 71], or rod-like, fig. 14c, with rotational disorder around one of the short axes [72, 73]. The first well-established example of a cubic phase composed of a packing of discrete inverse micelles is the phase Fd3m (= Q^^^). It has been observed in a variety of hydrated lipid mixtures, such as monoolein/oleic acid [29] and diglyceride/phosphatidylchoHne mixtures [74]. The structure has recently been solved [36] and is shown in fig. 15. As for the type I micellar cubic phase Pm3n, there are two types of aggregate in the unit cell. However, in the case of Fd3m both types of inverse micelle are quasi-spherical, but of different sizes. There are 8 of the larger and 16 of the smaller inverse micelles per unit cell. It is interesting to note that such a structure was predicted by a topological/geometrical study of the possible packings of fluid films [71]. Formation of the Fd3m cubic phase usually needs the presence of at least two lipid components, one of which is very weakly hydrophilic (e.g., fatty acid, diglyceride, etc). The explanation is probably that this permits a partial segregation of the two lipid components between the two types of micelle, with the less hydrophilic species locating preferentially in the smaller, more strongly negatively curved inverse micelles. A chiral cubic phase of spacegroup P4332 (= Q^^^) has been observed in a ternary lipid/protein/water system [29]. The proposed structure is derived from that of Ia3d: one water/lipid network remains, but the other is replaced by a network of quasispherical inverse micelles, within which the protein is located. The fact that this cubic phase is chiral has fascinating implications. In particular, although ordinary phospholipids appear not to be sufficiently chiral to form chiral cubic phases [76], more strongly chiral lipids might be found to do so.

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JM. Seddon and R.H. Tempter

IQ 3d (Q«^)

Pp3n)(Q''">

Imam (Qg»)

Fig. 13. Inverse bicontinuous cubic phases: a) Ia3d ( = Q^^O); b) Pn3m (= Q^^); c) Im3m ( = Q229) The structures for Ia3d and Pn3m are shown in their *rod-like' versions, which should correspond to low water contents. At higher water contents they will appear more like uniform thickness bilayers draped on the underlying minimal surface; cf., fig. Ic. Part of the underlying F-surface is shown for the Pn3m cubic phase. From [1].

Polymorphism of lipid-water systems

121

Fig. 14. The micellar cubic phase Pm3n ( = Q^^). The proposed clathrate/micelle structure (a) has now been ruled out. It is not yet established which of (b) or (c) is the correct structure. Also shown, between structures b and c, are electron density maps from the system sodium octanoate/p-xylene/water (taken from [29]). The left frame shows a section normal to the 3-fold axis through the point (1/4, 1/4, 1/4), and the right frame shows a section normal to the z-axis at z = 1/4. From [10].

J.M. Seddon and R.H. Templer

122

,-^

FcJ3nn

\

/

Fig. 15. The inverse micellar cubic phase Fd3m ( = Q^^"^). From [75].

5.8. Isotropic solution phases Although the translationally disordered solution phases such as micellar solutions, microemulsions, or so-called L3 (sponge) phases have so far been mainly associated with surfactant systems, we describe them briefly here, since it is likely that lipid systems in the presence of oil and/or naturally occurring co-surfactants may exhibit analogous behaviour. Short chain phospholipids (typically Ce or Cg) and lyso-phospholipids form micellar solutions at fairly high dilutions in water [3]. On the other hand, hydrated phospholipids can form inverse micellar solutions in the presence of certain organic solvents such as benzene [77]. Furthermore, at low hydrations in the presence of certain organic solvents such as alkanes, phosphatidylcholines form stiff, non-birefringent gels [78], whose structure appears to consist of entangled flexible inverse cylindrical micelles [79]. Inverse micellar solutions are also formed by phospholipids upon incorporation of large amounts of weakly polar amphiphiles such as diglycerides [74]. Microemulsions are isotropic solutions formed by amphiphile/oil/water mixtures [80]. The microstructure can consist of discrete micelles or inverse micelles, but when the volume fractions of oil and water are similar, bicontinuous structures tend to form, with the amphiphile forming a monolayer arranged as a random, connected porous interface between the oil and the water regions. The interface is believed to correspond to thermally disordered minimal surfaces [19, 20]. In some microemulsion systems, stiff gels are formed, which have been found to have a cubic phase structure. Unlike the bicontinuous lipid cubic phases, the structure appears to be

Polymorphism of lipid'Water systems

123

Fig. 16. Structure of the L3 'sponge' phase. The disordered interface may consist of either a bilayer, or an inverse bilayer. From [84].

based on a monolayer rather than a bilayer [44]. However, such monolayer cubic phases might be formed by phospholipids in the presence of organic solvents; work carried out recently in our laboratory hints at this. Certain surfactant systems form highly swollen lamellar phases, which may transform upon dilution to a so-called L3 or sponge phase [81-83]. This phase is essentially a disordered version of the bicontinuous cubic phases: the interface is highly flexible and thermal excitations break down the long range order of the network of channels so that the interface is no longer arranged on a lattice, see fig. 16. Such a sponge phase might occur in phospholipid systems in the presence of co-surfactants such as pentanol, which should drastically lower the rigidity of the lipid bilayer, thereby enhancing thermally-driven fluctuations. 4. Phase behaviour 4,1. Lyotropic phase diagrams Water content and temperature are the primary system variables for binary lipid/water systems. A hypothetical binary lipid/water phase diagram in which the transitions are driven predominantly by the former, is shown in fig. 17. There is a 'natural' sequence in which the various possiblefluidphases occur, determined by the average mean curvature of the polar-nonpolar interface [1, 20, 23, 85]. Although the phase diagrams of some surfactants show a striking similarity to regions of the hypothetical diagram, phospholipid phase diagrams invariably show a dependence on temperature as well as hydration. For example, fig. 18 shows the binary phase diagrams in water for the C16 and C12 saturated ether-linked phosphatidylethanolamines. At high

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JM. Seddon and R.H. Templer

0

4^ 'I

A^^N/V^NA/^A 9

a E

|2 Inverse Micellar Solution

JJ 0

H

Micellar Solution

I Water content (%)

100

Fig. 17. Hypothetical lipid/water binary phase diagram, where the transitions are driven by varying the water content. Regions denoted a, b, c and d contain intermediate phases, many of which are cubic. From [1].

temperatures, this class of phospholipid tends to adopt the Hn phase over a very wide range of water contents. Lowering the chainlength causes the appearance of the bicontinuous inverse cubic phases Ia3d, Im3m and Pn3m between the LQ and Hii phases. Curiously, some systems exhibit phase sequences which are not in accord with fig. 17. For example, in monoolein, the inverse bicontinuous cubic phases occur on the high water side of the lamellar phase [21, 86], even though they are inverse. It is also possible for an inverse bicontinuous cubic phase such as Ia3d to occur on the low hydration side of an inverse hexagonal Hn phase. An extensive compilation of binary and ternary phase diagrams has been given by Ekwall [87], and overviews of phase diagrams of lipid mixtures have been presented [13, 88, 89]. In addition to true binary phospholipid phase diagrams (i.e. lipid/water), it is common in the literature to find the term *binary' used to refer to binary lipid/lipid mixtures in the presence of an excess water phase. For various such binary lipid mixtures, a range of types of phase diagram are observed, from perfect mixing to eutectic, peritectic or monotectic behaviour. Generally speaking, deviations from ideal mixing become stronger when the lipids differ strongly in chainlength or headgroup type. A compilation of lipid phase diagrams has been published [90], and databases of lipid transition temperatures and enthalpies, and of phase diagrams, are currently being assembled [91]. A large number of reviews of

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Polymorphism of lipid-water systems

UJ

^HiO

(mol/fflol)

^H20

(moI/mo1)

b)DDPE

Fig. 18. Binary phase diagram of the phospholipid system: a) dihexadecyl-phosphatidylethanolamine/ water; b) didodecyl-phosphatidylethanolamine/water. For this system upon cooling, the La phase is metastable down to 35 °C, and then transforms to a metastable L^ gel phase. From [76].

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126

lipid phase transitions are available [1, 2, 7-10, 55, 92-100], and these should be consulted for much detail which will not be covered here. 4.2. Phase stability The factors responsible for controlling phase stability may be broken down into two types, which are interrelated. Firstly, the transverse interactions between adjacent lipid layers (Van der Waals, hydration,fluctuation,electrostatic) play an important role in stabilizing the structures [13, 101, 102]. In particular, for inverse non-lamellar phases the layer separations (hence lattice parameters) and interfacial curvatures are strongly coupled together. This might provide a mechanism for limiting the shrinking of bicontinuous phases. Secondly, lateral interactions within a lipid bilayer modulate the preferred interfacial area per molecule. There is a balance between the hydrophobic effect and any attractive interactions (e.g., hydrogen-bonding), tending to minimize the interfacial area, and repulsive chain and headgroup interactions, tending to expand it; see fig. 19. However, the various interactions occur at different depths within the lipid layer, and this may lead to a tendency for bending, either towards cylindrical or saddle-like surfaces (of negative Gaussian curvature). The detailed form of the bilayer stress profile is thus of great importance in determining the lipid polymorphism; see below. 4.3. Packing geometry and frustration Generally speaking, increasing the temperature will introduce more conformational disorder into the hydrocarbon chains, which will tend to expand the interfacial area per molecule. Conversely, increasing the water content tends to increase the lateral repulsions between the headgroups. This also increases the interfacial area per

HMd*9foup presstire Interfacial p

Chain

4.0 nm 1 -

Fig. 19. Balance of lateral interactions across a lipid bilayer. From [103].

Polymorphism of lipid-water systems

127

Fig. 20. Frustration in bilayer packing. Adapted from [25].

molecule, and hence also the extent of water-hydrocarbon contact. However, this expansion forces the chains to deviate away from their preferred conformational state, which costs energy, and leads to a tendency for each monolayer to curve towards its chain region. The *stiffness' of the bilayer to changes in area is given by the area compressibility modulus KA, which for phospholipids in the LQ phase typically has values in the region of 140 mN/m [13]. Within a certain temperature range, such an expansion of area does indeed occur. However, this forces the headgroups further apart than their optimal separation (usually any repulsive inter-headgroup terms will be less affected by temperature than those in the chain region), increasing the extent of water-hydrocarbon contact, which is disfavoured by the hydrophobic effect. Increased disorder of the chains could be accommodated without an expansion of the interface if each monolayer were to curve towards the water region outside it; see fig. 20. However, this would open up voids in the centre of the hydrocarbon chain region which, in the absence of any non-polar solutes, would be energetically prohibitively expensive. Thus in general within a flat lipid bilayer a state of physical frustration will often exist, whereby the compromise equilibrium interfacial area per molecule fully satisfies the packing preference of neither the headgroups nor the chains. For many lipids the La phase becomes unstable upon heating when the area per molecule exceeds a critical value (which can be as low as 60 A^ for phosphatidylethanolamines), and a transition to an inverse non-lamellar phase occurs. In transforming to an inverse phase, one possibility is for each lipid monolayer to curl right round into an inverse cylinder, these then packing onto a 2D hexagonal lattice as an Hn phase; see fig. 1. However, as is clear from fig. 21, there still remains a frustration in the chain packing in that all of the hydrophobic regions must be filled at a uniform liquid alkane density, but in order to fill the triangular regions in the centre (shaded), some of the chains must stretch away from their optimal conformational state. This problem might be partially alleviated by the interfaces of the lipid/water cylinders deforming away from a circular, towards a hexagonal cross-section [104]. Surprisingly, a way does exist for each lipid monolayer to develop a net mean curvature (splay) towards the water, yet without creating potential voids (and hence chain packing problems) within the hydrocarbon region. As may be seen from figs 3

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JM Seddon and R.H. Templet

Fig. 21. Packing frustration in the Hu phase. From [1].

and 5, if the bilayer deforms onto a saddle surface (of negative Gaussian curvature), this largely solves the problem, since the ends of the chains of each monolayer meet on the saddle surface without any voids, whilst since the area decreases on moving away in either direction from the saddle surface, each monolayer has developed a negative mean curvature (i.e. towards the water). By symmetry, since the two monolayers are equivalent, the saddle surface should have zero mean curvature, i.e. should be a minimal surface. Extending the surface indefinitely through space to form an infinite periodic minimal surface such as the P-surface; see fig. 4, then leads to the formation of an inverse bicontinuous cubic phase such as Im3m; see fig. 13. However, draping a bilayer onto an infinite periodic minimal surface does not provide a complete relief of the packing/curvature frustration. Since the Gaussian curvature of any infinite periodic minimal surface is not constant along the surface (it is most negative at the saddle points and rises to zero at the apices; see fig. 3), a constant thickness bilayer would not have a uniform (negative) monolayer mean curvature at the polar non-polar interfaces. Conversely, a uniform curvature would require a non-uniform thickness. Although both of these situations raise the energy of the system, a theoretical analysis [28] has shown that cubic phases can have a smaller amount of frustration than neighbouring lamellar and Hn phases, and can thus be expected to occur - as observed experimentally - between these two phases.

Polymorphism of lipid-water systems

129

4.4. Curvature elastic energy For large scale single bilayers, or for highly swollen systems, where the layer thickness is negligible compared with the dimensions of the bilayer or the layer spacing, the bilayer may be regarded as a thin elastic sheet. The energy cost associated with altering the mean or Gaussian curvature of this sheet is then given by the values, K and «G, of the mean and Gaussian curvature elastic moduli. In this regime, thermally excited (entropic) fluctuations may drive structural or phase transitions, at an elastic energy cost determined by the two curvature elastic moduli. However, for many biological lipid systems, the phases formed have relatively low hydrations (15-40 wt%), and the layer thickness is then comparable to the layer spacing or lattice parameter of the phase. In this regime, the stress profile across the layer needs to be taken into account in assessing the two curvature elastic moduli. This profile is liable to vary strongly with physico-chemical conditions such as temperature, hydration, pH, salt concentration, etc, and thus phase transitions in this regime might be driven by changes to the two curvature elastic moduli. In the treatment of Helfrich, the curvature elastic energy per unit area of a monolayer is given [105] by ^curv = (l/2)/c'"(Ci + C2 - Cof + /cgCcpCi)

(4)

where c\, C2 are the principal curvatures and CQ the spontaneous curvature of the monolayer (taken to be negative for curvature towards the water region), and H and K are the mean and Gaussian interfacial curvatures. The parameter K^ is the monolayer mean curvature elastic modulus, with a value in the region of 2 x 10"^^ J for typical phospholipids. This first term, which is quadratic in H, expresses the energy cost of deforming the monolayer away from the equilibrium mean curvature HQ = co/2. The second term gives the contribution to the free energy of the Gaussian curvature of the monolayer. For a bilayer, the value is not simply doubled, but is to first order given [106] by

4 = 2(AcS-2/c%t),

(5)

where t is the distance of the neutral surface (i.e. the surface of constant area under bending) of either monolayer from the mid-surface of the bilayer under cylindrical curvature. This term is zero for parabohc surfaces such as planes or cylinders, and thus is not relevant to the case of (flat) lamellar or cylindrical phases. On the other hand, for elliptic or hyperbolic surfaces the Gaussian term may be important, depending on the magnitude of /c^. However, for lipid systems not only the magnitude, but also the sign of i<^ is not well known, and this could have either the same, or the opposite sign of that for the monolayer «;g . Negative «^ should tend to favour elliptic surfaces such as bilayer vesicles. Conversely, positive values of KQ should tend to favour formation of inverse bicontinuous phases based on saddle surfaces. Note

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JM. Seddon and R.H. Tempter

that systems could in principle adopt a cubic phase against an unfavourable negative Gaussian curvature modulus, if there is some other favourable free energy term (e.g., entropy) which outweighs it. However, for this to occur, /c^ would probably have to be small in magnitude. For ternary surfactant/oil/water systems, it has been argued that the favoured phase is determined largely by the preferred value of interfacial mean curvature, in conjunction with packing constraints induced by the composition and geometry (Strom and Anderson, 1992). Although in such ternary systems the Gaussian curvature energy may play a minor role, this is not necessarily the case in lipid systems. A recent study of glycolipid inverse bicontinuous cubic phasesfindsthat /eg is comparable in magnitude to K^, and is of negative sign [107]. In the original Helfrich equation for the curvature energy there is an inherent instability due to the linear dependence on Gaussian curvature K, which is physically unrealistic: for a structure of positive K^, based on minimal surfaces, it predicts that the system could lower its free energy by allowing K to become increasingly negative. This would lead to structures which shrink indefinitely, or which are of indefinitely high genus. In reahty, such phases are frequendy stabilized at lattice parameters in the region of 100 A, and with relatively low genus values, such as 3 (per translation cell) for the cubic phases Ia3d, Im3m and Pn3m. One source of stabilization can arise from inclusion of higher order terms in the expression for the curvature elastic energy [106, 108-110]. Inclusion of a term quadratic in K leads to an equilibrium size for the unit cell, and implies a preferred value KQ of the bilayer Gaussian curvature. However, it is actually impossible for i^ to be uniform in a bicontinuous phase, since the underlying minimal surfaces must always containflatpoints (where K tends to zero) for the structure to be periodic. The genus 3 bicontinuous cubic phases appear to have the smallest relative variation in K along the interface, and should therefore tend to be preferred [106]. However, the three genus 3 cubic phases all have the same variance in K since they are related by the Bonnet transformation, and so other factors must determine which of these phases is the energetically favoured one. The most striking difference is the dimensionless interfacial area a = A/V^/^, which has the values of 2.3451, 2.4177 and 2.4533 for the P, F and G surfaces, respectively. This suggests that the phase sequence upon water dilution should be in the order G-F-P in order to accommodate the increased volume of the water, assuming the interfacial area per molecule remains constant. Although the limited data available to date suggests that this sequence is indeed the natural one in lipid systems, the situation in general is more complicated since the area per molecule is frequently a strong function of the water content. 4.5, Lateral stress profile Helfrich has shown that the mean and Gaussian curvature moduli K and KQ are directly related, respectively, to the first moment of the stress profile across the lipid monolayer, and the second moment of the stress profile across the bilayer [105]. The expected form of the stress profile t{z) for a lipid monolayer is shown infig.22. The lateral pressure in the chain region is balanced by the residual interfacial tension at

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Polymorphism of lipid-water systems

Fig. 22. Schematic lateral stress profile t(z) across a lipid monolayer of thickness d. From [1].

the polar/non-polar interface. There will also be a repulsive (or possible sometimes even attractive) lateral interaction acting between the polar headgroups. At the equilibrium area per molecule, the net lateral tension a, i.e. the integral of the stress profile across the monolayer, must equal zero:

"I

t(z)dz=^0.

(6)

However, in general (as previously discussed) there will be a tendency for the monolayer to curve, either towards the water region or towards the hydrocarbon chain region, depending on whether the interfacial tension is balanced primarily by the chain pressure or the headgroup pressure, respectively. This lateral torque tension r is given by the first moment of the stress profile of the flat monolayer, which is directly related to the product of the spontaneous curvature CQ and the mean curvature modulus K:

= / ^ ' < z)dz=

-Acco,

(7)

i.e. in the absence of any applied force, the monolayer will only be flat spontaneously if Co is zero. This approach of Helfrich does not lead to a separation of «; and CQ. However, Szleifer and co-workers have shown that k may be obtained from the first moment of the variation of the stress profile with mean curvature [111]. For a symmetrical bilayer at its equilibrium area per molecule, the torque tension r is always zero by symmetry; see fig. 23. However, the second moment of the

y.M. Seddon and R.H. Templer

132

^nz)\ K G » 0

Fig. 23. Schematic bilayer stress profile t(z) and its first and second moments. The thickness of the hydrocarbon chain region is 2c, h is the thickness of the interfacial region, a is the net lateral tension, r is the torque tension, and KQ is the Gaussian curvature modulus. From [1].

133

Polymorphism of lipid-water systems

| L ; . I . ' . - . ' . I . ! - . . •.'.•'.••••' I

L.:!i;.'.:ivi.'- ; T ? f t ^ 1 1 1

1 1 ••

I ' l l i l l

K, {ff,y

Hg phott prteurtort (iflokage) ILA .(fusion)

itetrople or invtrttd Cubic Phostt Fig. 24. Proposed routes for inverse bicontinuous cubic and Hn phase formation, via 'inverted micellar intermediates' (IMI). For the former phase, the route is proposed also to proceed via so-called 'interlamellar attachments' (ILA). Pfu^n^ and k\{n^f are the rates of formation of interlamcUar attachments and Hn phase precursors, respectively. From [115].

bilayer stress profile, which may be equated with the Gaussian curvature modulus: KG=

/

zh{z)dz

(8)

will not in general be zero. For a bilayer where the interfacial tension is balanced primarily by the chain pressure, shown schematically in fig. 23, the second moment, and hence KQ, will

134

J.M. Seddon and R.H. Templer

in general be positive, if the origin is set at the centre of the bilayer (note that this surface certainly does not correspond to the neutral surface upon deforming a bilayer into a saddle surface: the area per molecule at the mid-plane must increase compared to the flat bilayer). Thus such a bilayer can lower its elastic energy by deforming onto a saddle-surface (minimal surface at the bilayer centre by symmetry), hence tending to favour the formation of inverse bicontinuous cubic phases. Calculations of K and KG show that they depend strongly on the average area per molecule and chainlength [111]. However, we believe that there is still a difficulty in defining where to set the origin in evaluating the integrals, since the surface of inextension for a bilayer must lie close to the polar/non-polar interface of each monolayer. The form of the stress profile, and hence the first and second moments, will be modified by alterations such as changing the lipid chainlength, the headgroup hydrophilicity (e.g., by methylation), or by changing the solution properties such as the pH or salt concentration. 4,6. Defects and epitaxiality in phase transitions It has frequently been suggested that transitions to non-lamellar phases occur via the formation of defects such as inverse micelles [94-96]. Siegel has developed a model for such transitions, whereby the first step involves the formation of such an *inverted micellar intermediate' between apposed bilayers [112-114]. Two subsequent outcomes are possible (in addition to reversion), depending on the particular lipid system, fig. 24. Either the inverted micellar intermediate can fuse with neighbouring ones to form rod-like inverse micelles, or it can fuse with the surrounding monolayers to form an *interlamellar attachment', a fusion channel between the two bilayers. The former outcome should lead to the formation of the Hn phase, whereas the latter should lead to the formation of inverse bicontinuous structures such as cubic phases. The similarity between a fusion channel between two bilayers as shown in fig. 25,

Fig. 25. Fusion channel between two bilayers. From [1].

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Polymorphism of lipid-water systems

and the local structure of the Im3m (= Q^^^) cubic phase shown in fig. 13c, is indeed striking. Time-resolved cryo-transmission microscopy appears to have captured interlamellar attachment formation and the subsequent assembly into a cubic phase [116]. Defects, possibly corresponding to pores, have indeed been observed in lamellar LQ phases of surfactant systems by X-ray and neutron scattering, particularly when close to phase boundaries [117-122], For phospholipids, however, the cost of forming such holes is expected to be much higher, and it is therefore possible that their density would be too low to detect by scattering. In order to study defects in lipid phases by such scattering experiments, it is necessary to obtain monodomain samples, and this has proved to be quite problematical, particularly for more complex structures such as cubic phases. On the other hand, for certain amphiphile systems such as the polyoxyethylene surfactants, monodomain cubic phases grow spontaneously [117, 123-127]. In addition to allowing the study of defects, such monodomains also permit the study of the epitaxial relationships that exist as one phase transforms into another [124, 126]. For the surfactant hexaethylene glycol mono-n-dodecyl ether (C^EOe), the crystallographic planes of the lamellar, Ia3d (Q^^^) cubic, and the Hi phases were found to be aligned as shown in fig. 26. The (211) planes are in fact the densest planes in the cubic phase. Similarly, well-defined epitaxial relationships have been demonstrated between the various fluid phases observed in the sodium dodecyl sulphate/water system [122].

(001)

La

Q,.

H,

Fig. 26. Epitaxial relationships between the lamellar La, type I cubic Qa (spacegroup Ia3d) and type I hexagonal HQ phases of a polyoxyethylene surfactant. The crystallographic sections and directions are denoted by round and square brackets respectively. From [124].

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J.M. Seddon and R.H. Templer

For lipid systems forming inverse phases, much less information is currently available. It is known that the La-Hn transition occurs with the lamellar (001) planes aligned with the (10) planes of the Hn phase [128-130]. For the system monooleoyl glycerol, which forms inverse bicontinuous cubic phases, the (001) planes of the lamellar phase were again found to be ahgned with the (211) planes of the Ia3d cubic phase [M. Rawiso and J. Charvolin, unpublished observations, 130], although the latter authors also observed occasional alignment with the (220) planes. 5. Factors affecting lyotropic transitions 5.7. Types of transition There are three main types of phase transition between translationally ordered lyotropic phases. Firstly, there are transitions between ordered lamellar phases, such as crystal-crystal, crystal-gel, and gel-gel (e.g., L/3/--P/3/). Secondly, there are chainmelting transitions where the lower temperature phase is always lamellar, whereas the higher temperature phase, which is at least partially fluid, need not be lamellar, and need not even be liquid-crystalHne (e.g., it could be a micellar solution). Thirdly, there are transitions where both of the phases are fluid. The transition involves a change of symmetry and/or topology, for example, lamellar-hexagonal, lamellarcubic, cubic-cubic, etc. Furthermore, in principle any of the translationally ordered fluid phases can have transitions to isotropic solution phases (micellar solutions or microemulsions). In addition to the usual intensive thermodynamic variables such as temperature and pressure, transitions may also be induced isothermally and isobarically by changes in hydration, pH, salt concentration etc. The sensitivity of a transition to a given perturbation should be proportional to the free energy shift induced by the perturbation, divided by the transition entropy [13, 131]. Since transitions between fluid phases invariably involve small enthalpy (and hence entropy) changes, such transitions tend to be very sensitive to perturbations. The stabiHty of lipid phases should in principle be affected by electric and magnetic fields. The effects of the latter are expected to be very weak since the diamagnetic susceptibility anisotropy of Hpids is usually very small (unlike for thermotropic liquid crystals). Electric fields are much more important, and can bring about membrane fusion [132, 133] possibly in part by inducing inverse structures to form in the fusion regions. Chirality, notwithstanding its paramount importance in Biology, seems to have surprisingly little effect on lipid phase structure or on phase transitions [134, 135]. However, it has recently been found that the chirality of a dialkyl glycolipid does affect the transitions to non-lamellar phases for these systems [136]. Hydrostatic pressure is of great importance in the membrane Biology of marine species, but also has many powerful effects on most membranes from other species, affecting anaesthesia, permeability, excitabiHty and synaptic transmission [137, 138]. However, remarkably little work has so far been carried out on the effects of pressure on lipid polymorphism, particularly on the fluid phases. The gel-fluid transition of

Polymorphism of Upid-water systems

13V

phospholipids increases linearly by approximately 0.02 °C per atm of applied pressure [139, 140]. Effects on increasing the La-Hn transition are roughly twice as large [141-143]. Perhaps the most striking result (predicted in [1]) is that inverse cubic phases can be induced to form between the La and Hn phase in certain lipid systems by application of pressure [144]. 5.2. Effect of lipid chemical structure 5.2.1. Hydrocarbon chains Increasing the Hpid chainlength or the number of chains per polar headgroup has the effect of strongly increasing the hydrophobicity, and of increasing the chain-chain interactions. This drastically lowers the cmc (critical micelle concentration), increases the chain-melting temperature (e.g., gel-fluid bilayer), and tends to favour the formation of inverse non-lamellar phases. The La-Hn transition temperature falls steeply with increasing chainlength [145, 146]. The published data for the chainlength dependence of both the chain-melting, and the La-Hn transition temperatures are well fit by the following expression [147, 148]: Tt = {AHinc/ASinc)[(n - no)/(n - n(,)],

(9)

where AHmc and ^Sinc are the incremental values per CH2 group of the transition enthalpy and entropy; n is the chainlength and no and TIQ are the chainlengths at which the transition enthalpy and entropy extrapolate to zero. The presence of cis- or trans-douhh bonds in the chains has the effect both of drastically lowering the gel-fiuid transition temperature (typically by approximately 60 °C for a cw-unsaturated bond) and of lowering any transitions to inverse nonlamellar phases. The effects depend strongly upon the position of the double bond along the chain, the maximal effect occurring close to the middle of the chain [149]. For diacyl phosphoHpids, increasing asymmetry between the lengths of the two chains has the effect of lowering the chain melting transition temperatures [150]. For sufficientiy asymmetric chains, interdigitated gel phases tend to be induced [151-153]. Phospholipid phase behaviour is sensitive to the type of linkage between the chain and the polar headgroup. Ether linkages tend to increase the gel-fluid transition temperature by 1-5 °C, can induce the formation of interdigitated gel phases, and drastically lower the transition temperatures from the fluid lamellar La phase to inverse non-lamellar phases [145, 148, 154-158]. 5.2.2. Headgroups The chemical structure of lipid headgroups plays a major role in determining the lipid polymorphism. Seemingly minor modifications, such as replacement of a single proton by a methyl group, can profoundly alter the phase behaviour [1, 2, 9]. The crucial underlying factor appears to be the effective polarity of the headgroups [159], although charge (coulombic) and steric effects also play a role. In addition to the intrinsic hydrophilicity of a lipid headgroup, the effective polarity depends upon a number of factors such as the accessibility of different headgroup moieties to water.

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the possibility for direct headgroup-headgroup bonding, which weakens the interaction between the headgroup and water, etc. The latter factor may be responsible for the striking differences between the polymorphism of phosphatidylethanolamines compared to that of phosphatidylcholines [1]. For the former lipid there is the possibility for direct hydrogen-bonding between the headgroups, which is strikingly apparent in the crystal structure [51, 54], and which may still partially occur in the mesophases [160]. For phosphatidylcholines on the other hand, this possibility does not exist, and the headgroups interact much more strongly with water. This increases the hydration of the phases, modifies their structures, lowers the gel-fluid transition temperature, and tends to prevent the appearance of inverse non- lamellar phases for this latter class of lipid. The ionic, zwitterionic or non-ionic nature of a lipid headgroup appears to play a secondary role for many biological lipids. Thus the charged lipid phosphatidylglycerol has a strikingly similar phase behaviour in excess water [56] to that of zwitterionic phosphatidylcholine [161]. However, one difference is that the presence of the net charge on the former headgroup leads to an electrostatic swelling of the water layers at high hydrations [50]. Similarly, the polymorphism of certain non-ionic dialkyl glycolipids [162-164] is quite similar to that of the zwitterionic phosphatidylethanolamines [1]. A striking feature of the glycolipid systems is that the phase behaviour is dependent on the stereochemistry of the polar headgroup region [136]. For phospholipids, on the other hand, chirality does not appear to have any striking effects on their polymorphism [134]. Although racemic DPPE has been observed to have a more stable dehydrated crystalline form than the L-isomer [165], such differences do not seem to extend to the fluid phases: no difference was observed in the cubic phase formed by racemic dimethyl-DHPE and its chiral 1,2-sn counterpart [76], 5.3. Lipid mixtures The phase behaviour of lipid mixtures is of interest since it may give valuable insight into the factors responsible for lyotropic phase stability. Transverse interactions such as the hydration force between bilayers may be disproportionately modified by forming lipid mixtures [166]. Lateral interactions may also be modified in a non-additive way, with a strong effect on phase stability. Phases of non-uniform interfacial curvature may become favoured by the possibility for partial lateral segregation of different lipid species into regions of different curvature. Regions of two-phase coexistence may become more extensive, and three-phase coexistence becomes possible. Novel effects may occur, such as the formation of phases which do not appear for purely binary lipid/water systems. A further reason for the study of such mixtures is that biological membranes normally contain a complex variety of lipids, and this is certainly not accidental but must be related to their function. Mixing lipids together alters both the tendency for monolayer curvature, and the packing stresses within the system, and this has large effects on the formation of non-lamellar phases [167]. Incorporating a *bilayer-forming' lipid with one which forms non-lamellar phases has the effect of decreasing the spontaneous monolayer

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curvature of the latter, tending to stabilize it in the lamellar phase. This is observed for unsaturated phosphatidylethanolamine (PE) systems upon incorporation of phosphatidylcholine (PC). However, for certain composition ranges, the mixtures adopt an intermediate curvature structure, forming bicontinuous cubic phases [168, 169]. 5.4. Solution effects In general, increasing hydration tends to lower the chain melting transition temperatures of lipids, by as much as 50 °C, although there are exceptions to this rule for some surfactant systems and for certain lipid mixtures. As discussed earlier, within the fluid phase region, increasing the water content tends to push the equiUbrium towards phases of less negative/more positive interfacial mean curvature. However, this rule is not always obeyed, for reasons which are not yet well understood. 5.5. Solute effects There are a number of ways in which solutes can modify the phase equilibria of hpids. In general, polar solutes cause an osmotic dehydration by competing with the lipid for interaction with water [55]. This tends to favour the formation of ordered phases (gel or crystalline), and may induce inverse non-lamellar phases to form. Furthermore, this effect can be enhanced if the solute binds to the lipid headgroups, displacing bound water molecules. For charged lipids, ion screening and binding occur, particularly for divalent or multivalent ions. This leads both to a reduction of electrostatic repulsion, and to dehydration effects. However, some strongly hydrophilic molecules such as certain halide or organic ions, or alcohols, actually enhance the interfacial hydration upon binding, and these solutes therefore have opposite effects on the lipid polymorphism. The effect of pH is complicated by a number of factors [170]. Firstly, the pK^ of ionizable groups at the suriface of lipid membranes is strongly shifted to higher pH values (by as much as 3 pH units) compared to the pK^ value for the isolated group in bulk solution [13, 171]. Secondly, the headgroup hydrophiHcity varies with the degree of ionization, and thus indirect yet important (or even dominant) hydration effects are invariably also present as well as the direct electrostatic effects. Thirdly, the strength of any interlipid hydrogen-bonding will depend strongly on the pH. The net effect however is that increasing pH almost always lowers the chain-melting transition temperature of lipids [170], and increases the transition temperatures to inverse non-lamellar phases [1]. Amphiphilic solutes span a wide range of compounds ranging from long chain fatty acids and alcohols, monoacylglycerols and diacylglycerols, to more rigid molecules such as cholesterol. They are frequently soluble in phospholipids up to molar ratios as high as 2 : 1, even when, like diacylglycerols, they are too weakly amphiphilic to form any lyotropic liquid crystalline phases on their own in water. Such molecules are very important in membrane biology because they have a range of activities such as fusion, anaesthesia and cell signalling. Although they all adsorb preferentially with their polar groups near the lipid headgroups, and their hydrophobic parts embedded

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within the hydrocarbon chain region, the effects they have vary widely, depending on their chemical structure. Incorporation of long chain fatty acids and alcohols into phospholipid bilayers such as PC or PG tends to broaden yet increase the gel-fluid transition temperatures [61, 172]. Typically, on reaching a molar ratio of 2 : 1, the transition sharpens as it reaches its maximum temperature, which is in the region of 20-25 °C higher than that of the pure phospholipid in water (this temperature is also frequently higher than that of the pure solute in water). Such stoichiometric mixtures usually melt directly from the gel phase to non-lamellar phases, either Hn [60, 62-64] or bicontinuous inverse cubic [76, 173]. Monoacylglycerols (monoglycerides) such as the fusogen monooleoyl glycerol tend to promote the formation of Hn and inverse bicontinuous cubic phases [174-177]. This is not altogether surprising since pure monoacylglycerols in water tend to adopt such inverse non-lamellar phases [21, 22, 86, 178]. Diacylglycerols also induce the formation of the Hn phase upon incorporation in phosphoHpids [179-185]. At high mole fractions in PC a cubic phase is formed [181] which has been identified as an inverse micellar cubic phase of space group Fd3m [36, 74, 75]. It is well known that cholesterol tends to smear out the gel-fluid transition of phospholipids, fluidizing the gel phase and ordering the lamellar LQ phase, leading to enhanced stability of fluid bilayers. However, it also tends to promote formation of inverse phases such as Hn, not only in *non-lamellar' phospholipids such as PE [186] but even in PCs, when the chains are polyunsaturated [187]. A non-polar solute may be defined as any molecule (or atom) which partitions preferentially into the hydrophobic interior of a lipid mesophase. Such solutes range from simple alkanes, aromatic compounds and inert gases, to certain anaesthetics, drugs, peptides and proteins, although strictly speaking the latter group will generally also contain limited extents of polar regions. Although phase equilibria in ternary surfactant/oil/water systems have been studied extensively for many years [87], it is only relatively recently that the effects of non-polar solutes on lipid polymorphism have been studied [167, 182, 188, 189, 190-196]. Non-polar molecules exert their effects by partitioning into the hydrocarbon regions of the lipid phase, increasing the tendency for splay of each monolayer towards the water (increased tendency for negative mean interfacial curvature of each monolayer). This tends to facilitate the formation of inverse phases. A further important effect is to reduce chain packing constraints by partitioning into the interstices within the hydrocarbon region. This tends to favour the formation of inverse phases such as Hn, where there is a significant degree of chain stress due to the necessity to fill the hydrophobic region at a uniform density. The effects are largest on systems with small spontaneous curvatures. For example, addition of 5% alkane to a 3/1 DOPE/DOPC mixture reduces the La-Hn transition temperature by as much as 55 °C [189]. Even phosphatidylcholines may adopt inverse phases in the presence of alkanes [190, 191, 193]. The effects of dual-solvent (water and alkane) stress on PC/PE mixtures has been studied in order to remove packing constraints both in the hydrophobic and in the aqueous regions of the phases [195]. The results confirm that

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such lipid systems minimize their curvature energy, even if this requires separation into two bulk phases. 5.6. Phase metastability The study of lipid polymorphism has been hampered by the fact that many of the lipid phases are metastable, reverting in times ranging from seconds to years to more stable, or to true equilibrium phases [197]. A further complication is that the lipid molecules may be chemically unstable over such long timescales. Incubation of fully hydrated gel phase PC at low temperature leads to the formation of more ordered *sub-ger phases [198-202]. These subgel phases contain significant amounts of water and appear to have a crystalline chain packing, although it is not quite clear whether adjacent bilayers are weakly or strongly coupled together. Upon heating, the sub-gel phase undergoes an endothermic transition to the gel phase (usually Lpi). For lipids such as PE, which are more weakly hydrated, the tendency for metastability is very strong, leading to anhydrous crystalline lamellar phases upon incubation [37, 165, 203-208]. The pattern of metastability in fully hydrated L-DLPE is shown in fig. 27, where it is seen that both of the crystalline forms (denoted /3i and (32) observed after incubation have chain melting temperatures higher than that of the gel-fluid phase transition. Thus not only is the gel phase metastable, but also the fluid lamellar L^ phase is metastable below 43 °C [37]. For racemic DL-DPPE, the dehydrated crystalline form is particularly stable, having a chain melting transition in water on the initial heating scan at 82°C [165]. This shows that chirality can be a very significant factor in controlling metastability. Many other lipid systems exhibit metastability, such as mixed chain PE's [209], headgroup-methylated PE's [210]; phosphatidy[glycerol [211] and diacyl and dialkyl glycolipids [162, 212, 213]. Furthermore, stoichiometric fatty acid/PC ( 2 : 1 ) mixtures also adopt a subgel phase on incubation of the L/3 gel phase at low temperatures [62, 63]. 5.7. Transition kinetics The kinetics of the chain-melting transition of lipids has been studied by a number of techniques [214-216]. The advent of synchrotron radiation sources has allowed time-resolved diffraction to be employed to study lipid phase transition kinetics [178, 217-225]. Lipid phase transitions are thought to proceed either by nucleation and growth mechanisms, by spinodal decomposition, or by martensitic-type transformations [224]. In many cases the rate of the transition may be limited by the speed at which water can redistribute, rather than the time required for the lipids to rearrange themselves [219]. The L^-La transition of PE is a simple reversible two-state process occurring on the millisecond timescale. However, for phospholipids such as PC, both the L/3'-P^' tilted gel-rippled gel transition and the P/j^-La gel-fluid transition exhibit more complex multicomponent kinetic behaviour, with relaxation times spanning the range of milliseconds to seconds. Furthermore, on cooling the transitions are much

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o

H10 <

Fig. 27. The pattern of metastability of the fully hydrated L^ gel and LQ fluid lamellar phases of DLPE. There are two stable dehydrated crystalline forms, denoted I3\ and 02- ^H is shown as the difference in enthalpy of the lower temperature phases relative to the fluid lamellar La phase. From [37].

slower, taking minutes or hours to reach completion. Transitions between more ordered lamellar phases may be slower still. Lamellar-Hn transitions appear to be reversible two-state processes, requiring 110 seconds for completion, regardless of whether the lamellar phase is fluid (L^) [219, 220] or gel (Lp) [63]. There is still some confusion about the precise sequence of events which occur during the LQ-HH transition, but it seems that the Hn phase appears within tens of milliseconds, probably initially with the same spacing as the lamellar phase, before swelling to the equilibrium spacing over some seconds [225]. However, for small temperature jumps (of the order of 4°C) the transition becomes much slower, requiring as much as one week or more for completion. To date there have been few studies of the kinetics of transitions involving cubic phases, although in some cases they may be very slow and exhibit considerable hysteresis. For monoacylglycerols, it was found that cubic-cubic transition times ranged from 0.5 sec to 30 minutes [178]. In the case of a lipid extract from the extreme thermoacidophile 5. solfataricus, it was found to undergo a nearly irreversible transition from the L^ phase to a Pn3m (= Q'^'^^) inverse cubic phase on heating [226].

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For the unsaturated phospholipid DOPE a cubic phase was induced to form by thermal cycling, which was then strongly metastable [227]. Similarly, N-methyl-DOPE only forms cubic phases between L^ and Hn when the sample is cooled very slowly from the Hn phase [194] or is heated from the lamellar phase at less than 1°C per hour [228]. 6. Biological implications 6.1. Non-lamellar phases in biology The possible biological implications of lipid polymorphism have been discussed by a number of authors [1, 8, 9, 29, 94-96, 229, 230]. A scheme showing some of the ways in which non-lamellar structures may be of relevance to biomembrane morphology and function is given in fig. 28. Although many biomembranes contain large amounts of lipids which have strong tendencies to form phases such as Hn, there are few examples where non-lamellar phases have been definitely identified in cells. However, a considerable body of evidence suggests that non-lamellar structures do form, for example in membranes of microsomes [231-233], mitochondria [234, 235], and in tight junctions between cells [236-238]. Periodically curved bilayer structures have been observed in the membranes and extracted lipids of the bacterium Streptomyces hygroscopus [239]. This structure is suggested to be related to a twodimensional periodic minimal surface. It is interesting to note that the skeletons of echinoderms such as sea urchins are porous crystalline stmctures, which appear to be based on periodic minimal surfaces [240]. For example, the coronal plates of Cidaris rugosa are single crystals, with a structure [241] which is strikingly similar to the lyotropic Im3m (= Q^^^) cubic phase formed by lipids. Furthermore, non-lamellar phases do seem to form in certain situations. For example, paracrystalline inclusions in retina have been shown to consist of domains of Hii phase [242]. The plasma membrane of the archaebacterium Sulfolobus solfataricus appears to be based on the inverse bicontinuous cubic phase Pn3m (= Q^^^) [243]. However, probably the best example of a non-lamellar phase in biology is the prolamellar body of etiolated chloroplasts, which consists of six-fold or four-fold interconnected tubular membrane structures [244-247], strikingly similar to the structure elements of the inverse bicontinuous cubic phases Im3m (= Q^^^) and Pn3m (= Q^'^^). It is interesting to note that the structures of certain membraneous organelles in cells, for example in endoplasmic reticulum, bear a quite striking similarity to the L3 sponge phase. It has been suggested that Hquid-crystaUine phases, possibly including cubic phases, play a role in the process of fat digestion in vivo. During this process, triglyceride is hydrolysed first to diacylglycerol plus fatty acid, then to monoacylglycerol plus two fatty acid molecules. Studies of phase equilibria of lipid mixtures similar to those found in the intestine found that liquid-crystalline phases, as well as an L2 inverse micellar solution were formed, and it was suggested that the latter phase may coexist with mixed micelles in the human intestine [248]. In other model experiments in vitro, it was observed that first a lamellar phase, then a viscous isotropic, presumably

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^

Fig. 28. Scheme showing some of the possible roles of non-lamellar structures in biomembrane morphology and function: (1) exocytosis/fiision; (2) interbilayer connection/tight junction; (3) ion permeability. From [95].

cubic, phase was formed as the reaction of lipase with triglyceride proceeded [249]. Subsequent freeze-fracture electron microscopy results did not however show any clear evidence for a cubic phase [250, 251]. Work in our laboratory on a similar model system has also failed to find any evidence for cubic phase formation. A mechanism for how inverse bicontinuous phases such as Pn3m (= Q^^^) might be involved in fat digestion has been proposed [29]. These phases have the important

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property that all reactants and products, whether polar, non-polar or amphiphilic can diffuse freely across the structure. As the lipolysis proceeds, it would be highly advantageous to form such porous structures, rather than impermeable layers such as in a lamellar phase. 6.2. Membrane fusion and cell signal transduction Membrane fusion is a very common event in cell membranes, which requires the transient and localized destabilization of the bilayer stmcture. It has often been suggested that non-lamellar phases have a role to play in the underlying molecular mechanism of this process [1, 96, 112-114, 252-255] and evidence is accumulating in support of this view [115]. It is likely that inverse micellar structures form at the fusion site, lowering the activation barrier for the process. The presence of lipids such as phosphatidylethanolamine, having a tendency to adopt inverse non-lamellar phases, should facilitate this process. Furthermore, as previously mentioned, there is a very close relationship [1, 26, 115] between the stmcture of a fusion channel, shown in fig. 25, and the structure of an inverse bicontinuous cubic phase such as Im3m, shown in fig. 13c. Such a channel constitutes a bilayer deformation of negative Gaussian curvature. Thus the factors which drive a pure lipid/water system to undergo lamellar-cubic transitions, and the mechanism of the transition, may be very similar to some of those involved in membrane fusion in cells. The activation of phospholipase C, and the subsequent production of diacylglycerol in membranes, is associated with transmembrane signal transduction in cells, via activation of protein kinase C [179, 256, 257], and may also be involved in membrane fusion [180, 185, 258]. It is a striking result that diacylglycerols are potent promoters of inverse non-lamellar phases in phosphohpid systems [180, 181, 259]. Although diacylglycerols are too weakly hydrophiUc to form any lyotropic mesophases in solution on their own, they can be incorporated in phospholipids such as phosphatidylcholine up to mole fractions in excess of 0.7, inducing the formation first of the Hn phase, and then a cubic phase, with increasing concentration [181]. This latter phase has been shown to be an inverse micellar cubic phase, of spacegroup Fd3m (= Q^^'^) [75]. An electron microscopy study of the effect of treatment of PC/PE/cholesterol bilayer membranes with phospholipiase C, has identified structures which appear to be similar or identical to this inverse micellar cubic phase [258]. 6.3. Homeostatic control of 'phase stability' It was suggested some years ago that cells exhibit 'homeoviscous adaptation', whereby they maintain the ^fluidity' of their membranes close to some value which is optimal for their function [260]. Alternatively it has been proposed that rather than 'fluidity', it is the 'phase stability' [261, 262] or 'intrinsic curvature' [230] of the membrane lipids which is carefully regulated. Elegant experiments with Pseudomonas fluorescens [261], Acholeplasma laidlawii [262, 263], and Clostridium butyricum [264] have built up quite convincing evidence that cells may indeed regulate their lipid phase behaviour.

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6.4. Bilayer stress profile and regulation of membrane protein activity For mechanical stability, the residual interfacial tension 7 that exists on either side of a lipid bilayer must be balanced by the sum of the lateral pressures TTCH in the chain region, and TTHG in the headgroup regions [103]. Although most real systems will have contributions from both regions, the relative contributions may be quite different for different lipids. Thus for example, a strongly hydrated lipid such as PC should have a relatively large TTHG, and hence a small TTCH* whereas a more weakly hydrophilic lipid such as PE should have a smaller TTHG and hence a larger TTCHThus the stress profile across a bilayer membrane may be modified by changing the lipid composition (or the state of the lipid headgroups, e.g., by proton or ion binding). This means that the distribution of lateral stresses at different depths within the bilayer will change and, apart from modifying the lipid phase behaviour, might affect the conformation and mobility of proteins (or other embedded molecules), thereby modulating their activity. 5.5. Protein/lipid mixtures Although most of the lyotropic liquid-crystalline phases studied to date have involved pure hydrated lipids, since real cell membranes invariably contain associated integral or peripheral proteins, it is of great interest to study their effect on lipid polymorphism [229, 265]. For example, the transmembrane channel former gramicidin has been shown to have a strong tendency to induce Hn phase formation in a wide range of phospholipid systems. However, in general the effects are, not surprisingly, rather complex: binding or incorporation of proteins sometimes stabilizes the lamellar phase, but sometimes induces non-lamellar Hn or cubic phases to form. For example, the peptide melittin induces Hn phase formation in the charged lipids phosphatidic acid and PG, but stabilizes the lamellar phase of PE, and causes micellization of PC. These results clearly indicate that a range of effects such as partial charge neutralization, headgroup dehydration, protein insertion, etc occur, and these depend sensitively on the chemical structure and physical state of the lipid. Particularly interesting behaviour has been found for hydrated cytochrome c/monoacylglycerol mixtures, where a cubic phase of spacegroup P4332 ( = Q^^^) was detected at low water content [29]. This cubic phase is derived from the inverse bicontinuous cubic phase laSd (= Q^^^), replacing one of the sets of lipid/water channels by a network of protein/lipid inverse micelles. The unique feature of this cubic phase is that it is chiral. 7. Open problems Apart from the many putative roles of lipid polymorphism in biomembrane structure and function, discussed earlier, and the many remaining problems in our detailed understanding of the properties of lipid bilayers, the most pressing unresolved issues in this area are as follows: There is still no accurate free energy model for the cubic phases, which can correctly predict the phase sequence and relative stability. The relative importance

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of the spontaneous curvature co, and the mean and Gaussian curvature elastic moduli K and KG in controlling lipid polymorphism are still not well understood. The role of KG is particularly unclear: its sign is not even well established, and might be different in different lipid systems. The structures of many cubic and intermediate phases are still unknown. In most cases progress is hampered by the fact that very few Bragg peaks are observed in the diffraction patterns. The possible existence of bicontinuous structures which are of genus higher than 3 and/or which are based on monolayers rather than bilayers requires further investigation. For cubic phases there appears to be a correlation between the sign of the interfacial mean curvature (i.e. normal or inverse phases), and which crystallographic spacegroups are observed. For example, amongst micellar cubics, Fd3m (= Q^^^) is inverse, whereas Pm3n (= Q'^'^^) is normal (oil-in-water). Similarly, amongst the bicontinuous phases, Pn3m (= Q^^^) and Im3m (= Q^^^) are usually inverse. On the other hand, Ia3d (= Q'^^^) is commonly observed either as a normal or an inverse cubic phase. It is a mystery whether these correlations have any physical significance. It is unknown why in some systems incorrect' phase sequences are sometimes observed. For example, monoolein transforms from a lamellar to an inverse cubic phase with increasing water content. It is unclear whether there is a clear distinction between the discontinuous (micellar) and the bicontinuous cubic phases, or whether phases exist which are partially discontinuous in one component. In principle, chiral lipids could form non- centrosymmetric (chiral) cubic phases. In practice, for lipid systems studied to date this does not occur, presumably because the molecular chiraHty is too weak in the hydrated state. Bicontinuous cubic phases offer a partial solution to the problem of allowing monolayers to adopt their preferred mean curvature, without requiring a strong variation in the conformational state of molecules in different regions of the phase. It is unclear therefore why they are not more common in excess water, since most lipid systems will tend to have a non-zero preferred monolayer mean curvature, and the cubic phase could adjust its size to achieve an interfacial mean curvature close to this value. The relationship between the macroscopic, elastic properties of lipid layers and the underlying microscopic interactions needs further clarification. The limits on the swelling of cubic and other 3D lyotropic phases need to be determined. The lower limit for Ia3d appears to correspond to zero water content, since this cubic phase is formed by anhydrous strontium soaps. For Pn3m and Im3m it appears to be much larger, usually in excess of 30 wt% water. The upper limit of swelling of inverse bicontinuous phases is unclear, although theoretical arguments suggest that there should be a limit in the region of 300 A, set by the size at which thermal excitations will destroy the long range order. Certain cubic phases are metastable, remaining stable for weeks or months after cooling down below the equilibrium transition temperature to another phase; conversely, some systems only adopt cubic phases after prolonged incubation or thermal

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a)

c)

Fig. 29. Possible mechanism for transitions between bicontinuous cubic phases by layer stretching, without requiring layer intersections. The skeletal graphs of (a) the P-surface, (b) the F-surface, and (c) the gyroid surface are shown. By stretching the 6-connected node of the P-surface along the body diagonal direction and adjusting the angles, the graph of the F-surface, with 4-connected nodes, can be obtained. Stretching these 4-connected nodes then generates the 3-connected nodes of the gyroid skeletal graph. From [266].

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cycling. These phenomena are presumably connected to the complicated topological transformations required at such phase transitions. The role and use of periodic minimal surfaces in analyzing lipid phase structure and stability will continue to develop, and will contribute towards developing an understanding of the mechanism of transitions between non-lamellar phases. It is conceivable that the bilayer within a cubic phase drapes itself not exactly on a minimal surface (zero mean curvature) but rather on a minimal energy surface. Although it is tempting to invoke Bonnet-like transformations as being involved in transitions between Ia3d, Pn3m and Im3m cubic phases, such a mechanism would require layer self-intersection (via fusion events) during the transition, and this may be unphysical. An alternative mechanism, shown in fig. 29, would involve a continuous stretching of the layers during the transition, changing the connectivity of the nodes, followed by a relaxation to the new equilibrium phase. Precisely how such a process might proceed is poorly understood. Much more information is needed on the epitaxial relationships across phase boundaries, particularly for inverse systems, and on the role of defects in lyotropic transitions, in order to resolve some of these problems. Finally, the effects of polar, non-polar and amphiphilic solutes on lipid phase behaviour are still relatively poorly understood. Much future work will be addressed to resolving these issues. Acknowledgements This work was supported by grants from the former SERC (U.K.), the Royal Society (London) and Imperial College, London References 1. Seddon, J.M., 1990, Structure of the inverted hexagonal (Hn) phase, and non-lamellar transitions of lipids, Biochim. Biophys. Acta 1031, 1-69. 2. Tate, M.W., E.F. Eikenberry, D.C. Turner, E. Shyamsunder and S.M. Gruner, 1991, Nonbilayer phases of membrane lipids, Chem. Phys. Lipids 57, 147-164. 3. Luzzati, V., 1968, X-ray diffraction studies of lipid-water systems, in: Biological Membranes, Vol. 1, ed. D. Chapman (Academic Press, London) pp. 71-123. 4. Shipley, G.G., 1973, Recent X-ray diffraction studies of biological membranes and membrane components, in: Biological Membranes, Vol. 2, eds D. Chapman and D.F.H. Wallach (Academic Press, London) pp. 1-89. 5. Charvolin, J. and A. Tardieu, 1978, Lyotropic liquid crystals: Structure and molecular motions, Solid State Phys. Suppl. 14, 209-257. 6. Tiddy, G.J.T., 1980, Surfactant-water liquid crystal phases, Phys. Rep. 57, 1-46. 7. Small, D.M., 1986, Handbook of Lipid Research, Vol. 4 (Plenum Press, New York). 8. Larsson, K., 1989, Cubic lipid-water phases: Structures and biomembrane aspects, J. Phys. Chemistry 93, 7304-7314. 9. Lindblom, G. and L. Rilfors, 1989, Cubic phases and isotropic structures formed by membrane lipids - possible biological relevance, Biochim. Biophys. Acta 988, 221-256. 10. Fontell, K., 1990, Cubic phases in surfactant and surfactant-like lipid systems, Colloid Polym. Sci. 268, 264-285. 11. Chemistry and Physics of Lipids (Special Issue), Vol. 57, 1990. 12. Dubois-Violette, E. and B. Pansu (eds), 1990, International Workshop on Geometry and Interfaces, in: J. Phys. (Paris) Colloq. C7, Vol. 51.

150

J,M. Seddon and R.H. Templer

13. Cevc, G. and D. Marsh, 1987, Phospholipid Bilayers: Physical Principles and Models (Wiley, New York). 14. Meunier, J., D. Langevin and N. Boccara (eds), 1987, Physics of Amphiphilic Layers (Springer, Berlin). 15. Lipowsky, R., D. Richter and K. Kremer (eds), 1992, The Structure and Conformation of Amphiphilic Membranes (Springer, Berlin). 16. Lipowsky, R., 1991, The conformation of membranes, Nature 349, 475-481. 17. Prost, J. and F. Rondolez, 1991, Structures in colloidal physical chemistry. Nature (Suppl.) 350, 11-23. 18. Andersson, S., S.T. Hyde, K. Larsson and S. Lidin, 1988, Minimal surfaces and structures: From inorganic and metal crystals to cell membranes and biopolymers, Chem. Rev. 88, 221-242. 19. Scriven, L.E., 1976, Equilibrium bicontinuous structure, Nature 263, 123-125. 20. Scriven, L.E., 1977, Equilibrium bicontinuous structures, in: Micellization, Solubilization, and Microemulsions, Vol. 2, ed. K.L. Mittal (Plenum, New York) pp. 877-893. 21. Hyde, S.T., S. Andersson, B. Ericsson and K. Larsson, 1984, A cubic structure consisting of a lipid bilayer forming an infinite periodic minimal surface of the gyroid type in the glycerolmonooleatwater system, Z. Krystallogr. 168, 213-219. 22. Longley, W. and T.J. Mcintosh, 1983, A bicontinuous tetrahedral structure in a liquid-crystalline lipid, Nature 303, 612-614. 23. Charvolin, J., 1985, Crystals of interfaces: the cubic phases of amphiphile/water systems, J. Phys. (Paris) Colloq. C3 46, 173-190. 24. Mackay, A.L., 1985, Periodic minimal surfaces, Nature 314, 604-606. 25. Sadoc, J.F. and J. Charvolin, 1986, Frustration in bilayers and topologies of liquid crystals of amphiphilic molecules, J. Phys. (Paris) 47, 683-691. 26. Helfrich, W. and W. Harbich, 1987, Equilibrium configurations of fluid membranes, in: Physics of Amphiphilic Layers, eds J. Meunier, D. Langevin and N. Boccara (Springer, Beriin) pp. 58-63. 27. Charvolin, J. and J.F. Sadoc, 1987, Periodic systems of fhistrated fluid films and bicontinuous cubic structures in liquid crystals, J. Phys. France 48, 1559-1569. 28. Anderson, D.M., S.M. Gruner and S. Leibler, 1988, Geometric aspects of the frustration in the cubic phases of lyotropic liquid crystals, Proc. Nat. Acad. Sci. USA 85, 5364-5368. 29. Mariani, R, V. Luzzati and H. Delacroix, 1988, Cubic phases of lipid-containing systems. Structure analysis and biological imphcations, J. Mol. Biol. 204, 165-189. 30. Hyde, S.T., 1989, Microsmicture of bicontinuous surfactant aggregates, J. Phys. Chem. 93, 14581463. 31. Bouligand, Y., 1990, Comparative geometry of cytomembranes and water-lipid systems, J. Phys. (Paris) 51(C7), 35-52. 32. Gulik-Krzywicki, T., L.P. Aggerbeck and K. Larsson, 1984, The use of freeze-fracture and freezeetching electron microscopy for phase analysis and structure determination of lipid systems, in: Surfactants in Solution, Vol. 1, eds K. Mittal and B. Lindman (Plenum, New York) pp. 237-257. 33. International Tables for Crystallography, 1983, Vol. A, ed. T. Hahn (D. Reidel, Dordrecht). 34. Franks, N.P. and Y.K. Levine, 1981, Low-angle X-ray diffraction, in: Membrane Spectroscopy, ed. E. Grell (Springer, Beriin) pp. 437-487. 35. Zaccai, G. and B. Jacrot, 1983, Small angle neutron scattering, Annu. Rev. Biophys. Bioeng. 12, 139-157. 36. Luzzati, V., R. Vargas, A. Gulik, P. Mariani, J. M. Seddon and E. Rivas, 1992, Lipid polymorphism: A correction. The structure of the cubic phase of extinction symbol Fd- consists of two types of disjointed reverse micelles embedded in a 3D hydrocarbon matrix. Biochemistry 31, 279-285. 37. Seddon, J.M., K. Harlos and D. Marsh, 1983, Metastability and polymorphism in the gel and fluid bilayer phases of dilauroyl phosphatidylethanolamine, J. Biol. Chem. 258, 3580-3854. 38. Schoen, A.H., 1970, Infinite periodic minimal surfaces without self-intersections, NASA Technical Note TD-5541. 39. Hyde, S.T., 1990, Curvature and the global structure of interfaces in surfactant-water systems, J. Phys. (France) 51(C7), 209-228.

Polymorphism of lipid-water systems

J 51

40. Anderson, D.M., H.T. Davis, L.E. Scriven and J.C.C. Nitsche, 1990, Periodic surfaces of prescribed mean curvature, Adv. Chem. Phys. 77, 337-396. 41. Barois, P, S.T. Hyde, B.W. Ninham and T. Dowling, 1990, Observation of tvi^o phases within the cubic phase region of a ternary surfactant, Langmuir 6, 1136-1140. 42. Strom, P and D.M. Anderson, 1992, The cubic phase region in the system didodecylmethylammonium bromide-water-styrene, Langmuir 8, 691-709. 43. K6kicheff, P and B. Cabane, 1987, Betv^^een cylinders and bilayers: structures and intermediate mesophases of the SDS/water system, J. Phys. (Paris) 48, 1571-1583. 44. Radiman, S., C. Toprakcioglu and A.R. Faruqi, 1990, Symmetry transition in the cubic phase of a ternary surfactant system, J. Phys. France 51, 1501-1508. 45. Pascher, I., M. Lundmark, P.-G. Nyholm and S. Sundell, 1992, Crystal structures of membrane lipids, Biochim. Biophys. Acta 1113, 339-373. 46. Blaurock, A.E. and T.J. Mcintosh, 1986, Structure of the crystalline bilayer in the subgel phase of phosphatidylglycerol. Biochemistry 25, 299-305. 47. Tardieu, A., V. Luzzati and F.C. Reman, 1973, Structure and polymorphiam of the hydrocarbon chains of Hpids: A study of lecithin-water phases, J. Mol. Biol. 75, 711-733. 48. Ranck, J.L., T. Keira and V. Luzzati, 1977, A novel packing of the hydrocarbon chains in lipids: The low temperature phases of dipalmitoyl phosphatidylglycerol, Biochim. Biophys. Acta 488, 432-441. 49. Doucet, J., A.M. Levelut and M. Lambert, 1983, X-ray study of single domains of 1,2-dipalmitoylsn-phosphatidylcholine with less than 5% water. Acta Crystallogr. B39, 724-731. 50. Vargas, R., P. Mariani, A. Gulik and V. Luzzati, 1992, The cubic phases of lipid-containing systems. The structure of phase Q^^^ (space group Pm3n): An X-ray scattering study, J. Mol. Biol. 225, 137-145. 51. Elder, M., P. Hitchcock, R. Mason and G.G. Shipley, 1977, A refinement analysis of the crystallography of the phospholipid, 1,2-dilauroyl-DL-phosphatidylethanolamine, and some remarks on lipid-lipid and lipid-protein interactions, Proc. R. Soc. London Ser. A 354, 157-170. 52. Pearson, R.H. and I. Pascher, 1979, The molecular structure of lecithin dihydrate. Nature 281, 499-501. 53. Pascher, I. and S. Sundell, 1986, Membrane lipids: Preferred conformational states and their interplay. The crystal strucure of dilauroylphosphatidyl-N,N- dimethylethanolamine, Biochim. Biophys. Acta 855, 68-78. 54. Hauser, H., I. Pascher, I.H. Pearson and S. Sundell, 1981, Preferred conformation and molecular packing of phosphatidylethanolamine and phosphatidylcholine, Biochim. Biophys. Acta 650, 2151. 55. Cevc, G., 1991, Isothermal lipid phase transitions, Chem. Phys. Lipids 57, 293-307. 56. Watts, A., K. Harlos, W. Maschke and D. Marsh, 1978, Control of the structure and fluidity of phosphatidylglycerol bilayers by pH titration, Biochim. Biophys. Acta 510, 63-74. 57. StUmpel, J., K. Harlos and H. Eibl, 1980, Charge-induced pretransition in phosphatidylethanolamine multilayers: the occurrence of ripple structures, Biochim. Biophys. Acta 599, 464-472. 58. Harlos, K., J. Stumpel and H. Eibl, 1979, Influence of pH on phosphatidic acid multilayers: A rippled structure at high pH values, Biochim. Biophys. Acta 555, 409-416. 59. Safinya, C.R., D. Roux, G.S. Smith, S.K. Sinha, P Dimon, N.A. Clark and A.M. Bellocq, 1986, Steric interactions in a model multimembrane system: a synchrotron X-ray study, Phys. Rev. Lett. 57, 2718-2721. 60. Marsh, D. and J.M. Seddon, 1982, Gel-inverted hexagonal (La-Hn) phase transitions in phosphatidylethanolamines and fatty acid-phosphatidylcholine mixtures, demonstrated by ^^P NMR spectroscopy and X-ray diffraction, Biochim. Biophys. Acta 690, 117-123. 61. Boggs, J.M., G. Rangaraj and K.M. Koshy, 1986, Effect of hydrogen-bonding and non-hydrogen bonding long chain compounds on the phase transition temperatures of phospholipids, Chem. Phys. Lipids 40, 23-34. 62. Koynova, R.D., A.I. Boyanov and B.G. Tenchov, 1987, Gel-state metastability and nature of the azeotropic points in mixtures of saturated phosphatidylcholines and fatty acids, Biochim. Biophys. Acta 903, 186-196.

152

63.

64.

65.

66. 67. 68. 69. 70. 71. 72.

73. 74. 75. 76. 77. 78. 79.

80. 81. 82. 83. 84. 85. 86.

J.M. Seddon and R.H. Tempter

Koynova, R.D., B.G. Tenchov, P.J. Quinn and P. Laggner, 1988, Structure and phase behaviour of hydrated mixtures of L-dipalmitoylphosphatidylcholine and palmitic acid. Correlations between structural rearrangements, specific volume changes and endothermic events, Chem. Phys. Lipids 48, 205-214. Cevc, G., J.M. Seddon, R. Hartung and W. Eggert, 1988, Phosphatidylcholine-fatty acid membranes, I. Effects of protonation, salt concentration, temperature, and chain-length on the colloidal and phase properties of mixed vesicles, bilayers, and non-lamellar structures, Biophys. Acta 940, 219-240. Templer, R.H., K.H. Madan, N.A. Warrender and J.M. Seddon, 1992, Swollen lyotropic cubic phases in fiilly hydrated mixtures of monoolein, dioleoylphosphatidylcholine and dioleoylphosphatidylethanolamine, in: Structure and Conformation of Amphiphilic Membranes. Springer Proceedings in Physics, eds R. Lipowsky and D. Richter (Springer, Heidelberg) pp. 262-265. Bruinsma, R., 1992, Elasticity and excitations of minimal crystals, J. Phys. II France 2, 425-451. Luzzati, V. and PA. Spegt, 1967, Polymorphism of lipids. Nature 215, 701-704. Tardieu, A., 1972, PhD thesis. University de Paris-Sud. Tardieu, A. and V. Luzzati, 1970, Polymorphism of lipids. A novel cubic phase: A cage-like network of rods with enclosed spherical micelles, Biophys. Acta 219, 11-17. Fontell, K., K.K. Fox and E. Hansson, 1985, On the structure of the cubic phase Ii in some lipid-water systems, Mol. Cryst. Liq. Cryst. Lett. 1, 9-17. Charvolin, J. and J.F. Sadoc, 1988, Periodic systems of fmstrated fluid films and micellar cubic structures in liquid crystals, J. Phys. (Paris) 49, 521-526. Eriksson, P.-O., L. Rilfors, G. Lindblom and G. Arvidson, 1985, Multicomponent spectra from ^^P NMR studies of the phase equilibria in the system dioleoylphosphatidylcholine-dioleoylphosphatidylethanolamine-water, Chem. Phys. Lipids 37, 357-371. Eriksson, P.-O., G. Lindblom and G. Arvidson, 1987, NMR studies of micellar aggregates in 1-acyl-sn-glycerophosphocholine systems. The formation of a cubic liquid crystalline phase, J. Phys. Chem. 91, 846-853. Seddon, J.M., 1990, An inverse face-centred cubic phase formed by diacylglycerol-phosphatidylcholine mixtures. Biochemistry 29, 7997-8002. Seddon, J.M., E.A. Bartle and J. Mingins, 1990, Inverse cubic liquid-crystalline phases of phospholipids and related lyotropic systems, J. Phys.: Condens Matter 2, SA285-SA290. Seddon, J.M., J.L. Hogan, N.A. Warrender and E. Pebay-Peyroula, 1990, Structural studies of phospholipid cubic phases. Prog. Colloid Polym. Sci. 81, 189-197. Luisi, PL., 1985, Enzymes hosted in reverse micelles in hydrocarbon solution, Angew. Chem. Int. Ed. Engl. 24, 439-528. Scartazzini, R. and PL. Luisi, 1988, Organogels from lecithins, J. Phys. Chem. 92, 829-833. Schurtenberger, P., L.J. Magid, J. Penfold and R. Heenan, 1990, Shear aligned lecithin reverse micelles: A small-angle neutron scattering study of the anomalous water-induced micellar growth, Langmuir 6, 1800-1803. de Gennes, P.G. and C. Taupin, 1982, Microemulsions and the flexibility of oil/water interfaces, J. Phys. Chem. 86, 2294-2304. Porte, G., J. Appel, P Bassereau and J. Marignan, 1989, LQ to L3: A topology driven transition in phases of infinite fluid membranes, J. Phys. France 50, 1355-1347. Gates, M.E. and D. Roux, 1990, Random bilayer phases of dilute surfactant solutions, J. Phys. Condens. Matter 2, SA339-SA346. Strey, R., R. SchomScker, D. Roux, F. Nallet and U. Olsson, 1990, Dilute lamellar and L3 phases in the binary water-C^Es system, J. Chem. Soc. Faraday Trans. 86, 2253-2261. Strey, R., J. Winkler and L. Magid, 1991, Small-angle neutron scattering from diffuse interfaces, 1. Mono- and bilayers in the water-octane-Ci2E5 system, J. Phys. Chem. 95, 7502-7507. Winsor, P.A., 1968, Binary and multicomponent solutions of amphiphilic compounds. Solubilization and the formation, structure, and theoretical significance of liquid crystalline solutions, Chem. Rev. 68, 1-40. Lutton, E.S., 1965, Phase behaviour of aqueous systems of monoglycerides, J. Am. Oil Chem. Soc. 42, 1068-1070.

Polymorphism of lipid-water systems

153

87. Ekwall, P., 1975, Composition, properties and structures of liquid crystalline phases in systems of amphiphilic compounds, Adv. Liq. Cryst. 1, 1-142. 88. Lee,-A.G., 1977, Lipid phase transitions and phase diagrams, 11. Mixtures involving lipids, Biochim. Biophys. Acta 472, 285-344. 89. Lee, A.G., 1978, Calculation of phase diagrams for non-ideal mixtures of lipids, and a possible nonrandom distribution of lipids in lipid mixtures in the liquid-crystalline phase, Biochim. Biophys. Acta 507, 433-444. 90. Marsh, D., 1987, Handbook of Lipid Bilayers (CRC Press, Boca Raton, FL). 91. Caffrey, M., D. Moynihan and J. Hogan, 1991, A database of lipid phase transition temperatures and enthalpy changes, Chem. Phys. Lipids 57, 275-291. 92. Lee, A.G., 1977, Lipid phase transitions and phase diagrams, I. Lipid phase transitions, Biochim. Biophys. Acta 472, 237-281. 93. Silvius, J.R., 1982, Lipid phase transitions, in: Lipid-Protein Interactions, Vol. 2, eds PC. Jost and O.H. Griffith (Wiley, New York) pp. 239-281. 94. Cullis, PR., M.J. Hope, B. de Kniijff, A.J. Verkleij and C.P Tilcock, 1985, Structural properties and functional roles of phospholipids in biological membranes, in: Phospholipids and Cellular Recognition, ed. J.F. Kuo (CRC Press, Boca Raton, FL) pp. 1-59. 95. Cullis, PR., M.J. Hope and C.PS. Tilcock, 1986, Lipid polymorphism and the roles of lipids in membranes, Chem. Phys. Lipids 40, 127-144. 96. de Kruijff, B., PR. Cullis, A.J. Verkleij, M.J. Hope, C.J.A. van Echteld and T.R Taraschi, 1985, Lipid polymorphism and membrane function, in: The Enzymes of Biological Membranes, 2nd edition, ed. A.N. Martinosi (Plenum, New York) pp. 131-204. 97. Tilcock, C.PS., 1986, Lipid polymorphism, Chem. Phys. Lipids 40, 109-125. 98. Gruner, S.M., 1989, Stability of lyotropic phases with curved interfaces, J. Phys. Chem. 93, 7562-7570. 99. Marsh, D., 1991, General features of phospholipid phase transitions, Chem. Phys. Lipids 57, 109120. 100. Seddon, J.M. and G. Cevc, 1993, Lipid polymorphism: Structure and stability of lyotropic mesophases of phospholipids, in: Phospholipids Handbook, ed. G. Cevc (Marcel Dekker, New York) pp. 403-454. 101. Rand, R.P and V.A. Parsegian, 1989, Hydration forces between phospholipid bilayers, Biochim. Biophys. Acta 988, 351-376. 102. Israelachvili, J.N., 1991, Intermolecular and Surface Forces, 2nd edition (Academic Press, London). 103. Israelachvili, J.N., S. Marcelja and R. Horn, 1980, Physical principles of membrane organization, Q. Rev. Biophys. 13, 121-200. 104. Nayaran, O., PT.C. So, D.C. Turner and S.M. Gruner, 1990, Volume constriction in a lipid-water liquid crystal using high pressure X-ray diffraction, Phys. Rev. A 42, 7479-7482. 105. Hclfrich, W., 1981, Amphiphilic mesophases made of defects, in: Physics of Defects, eds R. Balian, M. Kidman and J.P Poirier (North-Holland, Amsterdam) pp. 715-755. 106. Helfrich, W. and H. Rennschuh, 1990, Landau theory of the lamellar-to-cubic phase transition, J. Phys. (France) 51(C7), 189-195. 107. Turner, D.C, Z.G. Wang, S.M. Gruner, D.A. Mannock and R.N. McElhaney, 1992, A structural study of the inverted cubic phases of didodecyl-alkyl-b-D-glucopyranosyl-rac-glycerol, J. Physique II 2, 2039-2063. 108. Hyde, S.T., I.S. Barnes and B.W. Ninham, 1990, Curvature energy of surfactant interfaces confined to the plaquettes of a cubic lattice, Langmuir 6, 1055-1062. 109. Fogden, A., S.T. Hyde and G. Lundberg, 1991, Bending energy of surfactant films, J. Chem. Soc. Faraday Trans. 87, 949-955. 110. Ljunggren, S. and J.C. Eriksson, 1992, Minimal surfaces and Winsor II microemulsions, Langmuir 8, 1300-1306. 111. Szleifer, I., D, Kramer, A. Ben-Shaul, W.M. Gelbart and S.A Safran, 1990, Molecular theory of curvature elasticity in surfactant films, J. Chem. Phys. 92, 6800-6817. 112. Siegel, D.P, 1986, Inverted micellar intermediates and the transitions between lamellar, cubic and inverted hexagonal lipid phases, I. Mechanism of the La-Hji phase transitions, Biophys. J. 49, 1155-1170.

154

J.M. Seddon and R.H. Templer

113. Siegel, D.P., 1986, Inverted micellar intermediates and the transitions between lamellar, cubic and inverted hexagonal lipid phases, II. Implications for membrane-membrane interactions and membrane fusion, Biophys. J. 49, 1171-1183. 114. Siegel, D.P., 1986, Inverted micellar intermediates and the transitions between lamellar, cubic and inverted hexagonal amphiphile phases. III. Isotropic and inverted cubic state formation via intermediates in transitions between La-Hn phases, Chem. Phys. Lipids 42, 279-301. 115. Ellens, H., D.P Siegel, D. Alford, P.L. Yeagle, L. Boni, L.J. Lis, RJ. Quinn and J. Bentz, 1989, Membrane fusion and inverted phases. Biochemistry 28, 3692-3703. 116. Siegel, D.P., J.L. Bums, M.H. Chestnut and Y. Talmon, 1989, Intermediates in membrane fusion and bilayer/nonbilayer phase transitions imaged by time-resolved cryo-transmission electron microscopy, Biophys. J. 56, 161-169. 117. Ran9on, Y. and J. Charvolin, 1988, Fluctuations and phase transformations in a lyotropic liquid crystal, J. Phys. Chem. 92, 6339-6344. 118. Holmes, M.C. and J. Charvolin, 1984, Smectic-nematic transition in a lyotropic liquid crystal, J. Phys. Chem. 88, 810-818. 119. Boden, N., S.A. Come, M.C. Holmes, RH. Jackson, D. Parker and K.W. Jolley, 1986, Orderdisorder transitions in solutions of discoid micelles, J. Phys. (Paris) 47, 2135-2144. 120. K6kicheff, P., B. Cabane and M. Rawiso, 1984, Structural defects of a lamellar lyotropic mesophase: A neutron scattering study, J. Physique Lett. 45, 813-821. 121. Hendrikx, Y, J. Charvolin, P. Kakicheff and M. Roth, 1987, Structural fluctuations in the lamellar phase of sodium decyl sulphate / decanol / water, Liq. Cryst. 2, 677-687. 122. K6kicheff, P. and B. Cabane, 1988. Crystallography of systems with long periods: A neutron scattering study of sodium dodecyl sulphate / water mesophases, Acta Crystallogr. B44, 395-406. 123. Ran9on, Y and J. Charvolin, 1987, Displacement disorder in a liquid crystalline phase with cubic symmetry, J. Phys. (Paris) 48, 1067-1073. 124. Ran9on, Y and J. Charvolin, 1988, Epitaxial relationships during phase transformations in a lyotropic liquid crystal, J. Phys. Chem. 92, 2646-2651. 125. Clerc, M., A.M. Levelut and J.F. Sadoc, 1990, X-ray study of phase transitions in amphiphilic systems, J. Phys. (France) 51(C7), 97-104. 126. Clerc, M., A.M. Levelut and J.F. Sadoc, 1991, Transitions between mesophases involving cubic phases in the surfactant-water systems. Epitaxial relations and their consequences in a geometrical framework, J. Phys. 11 France 1, 1263-1276. 127. Sotta, R, 1991, Equilibrium shape of lyotropic cubic monocrystals, J. Phys. II France 1, 763-772. 128. Gruner, S.M., K.J. Rothschild and N.A. Clark, 1982, X-ray diffraction and electron microscope study of phase separation in rod outer segment photoreceptor membrane multilayers, Biophys. J. 39, 241-251. 129. Gruner, S.M., K.J. Rothschild, W.J. DeGrip and N.A. Clark, 1985, Co-existing lyotropic liquid crystals: Commensurate, faceted and co-planar single hexagonal (Hn) domains in lamellar photreceptor membranes, J. Phys. (Paris) 46, 193-201. 130. Templer, R.H., N.A. Warrendcr, R. Meadows and J.M. Seddon, 1992, Epitaxial relationships between adjacent phases in hydrated monoolein, in: Structure and Conformation of Amphiphilic Membranes. Springer Proceedings in Physics, eds R. Lipowsky and D. Richter (Springer, Heidelberg) pp. 230-233. 131. Trauble, H., M. Teubner, R Wooley and H. Eibl, 1976, Electrostatic interactions at charged lipid membranes, 1. Effects of pH and univalent cations on membrane structure, Biophys. Chem. 4, 319-346. 132. Zimmermann, U., 1982, Electric field-mediated fusion and related electrical phenomena, Biochim. Biophys. Acta 694, 227-277. 133. Zimmermann, U., 1983, Electrofiision of cells: Principles and industrial potential. Trends Biotechnol. 1, 149-155. 134. Amett, E.M., J.M. Gold, N. Harvey, E.A. Johnson and L.G. Whitesell, 1988, Stereoselective recognition in phospholipid monolayers, in: Biotechnological Applications of Lipid Microstructures, eds B.R Gaber, J.M. Schnur and D. Chapman (Plenum, New York) pp. 21-36.

Polymorphism of lipid-water systems

155

135. Andelman, D., 1989, Chiral discrimination and phase transitions in Langmuir monolayers, J. Am. Chem. Soc. Ill, 6536-^544. 136. Mannock, D.A., R.N.A.H. Lewis, R.N. McElhaney, M. Akiyama, H. Yamada, D.C. Turner and S.M. Gruner, 1992, The effect of the chirality of the glycerol backbone on the bilayer and nonbilayer phase transitions in the diastereomers of di-dodecyl-D-glucopyranosyl glycerol, Biophys. J. 63, 1355-1368. 137. Uver, M.J., K.W. Miller, W.D.M. Paton and E.B. Smith, 1971, Pressure reversal of anaesthesia. Nature 231, 368-371. 138. MacDonald, A.G., 1984, The effects of pressure on the molecular structure and physiological functions of cell membranes, Philos. Trans. R. Soc. London B304, 47-68. 139. Liu, N.L and R.L. Kay, 1977, Redetermination of the pressure dependence of the lipid bilayer phase transition. Biochemistry 16, 3484-3486. 140. Wong, P.T.T., 1986, Phase behaviour of phospholipid membranes under high pressure, Physica 139 and 140B, 847-852. 141. Yager, P. and E.L. Chang, 1983, Destabilization of a lipid non-bilayer phase by high pressure, Biochim. Biophys. Acta 731, 491-494. 142. Chang, E.L. and P. Yager, 1983, Effect of high pressure on a lipid non-bilayer phase, Mol. Cryst. Liq. Cryst. 98, 125-129. 143. Shyamsunder, E., P. Botos and S.M. Gruner, 1989, Comment on: X-ray diffraction study of the effects of pressure on bilayer to nonbilayer lipid membrane phase transitions, J. Chem. Phys. 90, 1293-1295. 144. So, PT.C, 1992, PhD thesis, Princeton University. 145. Seddon, J.M., G. Cevc, R.D. Kaye and D. Marsh, 1984, X-ray diffraction study of the polymorphism of hydrated diacyl and dialkyl phosphatidylethanolamines. Biochemistry 23, 2634-2644. 146. Lewis, R.N.A.H., D.A. Mannock, R.N. McElhaney, D.C. Turner and S.M. Gruner, 1989, Effect of fatty acyl chain length and structure on the lamellar gel to liquid-crystalline and lamellar to reversed hexagonal phase transitions of aqueous phosphatidylethanolamine dispersions. Biochemistry 28, 541-548. 147. Marsh, D., 1991, Analysis of the chainlcngth dependence of lipid phase transition temperatures: Main and pretransitions of phosphatidylcholines; main and non-lamellar transitions of phosphatidylethanolamines, Biochim. Biophys. Acta 1062, 1-6. 148. Seddon, J.M., G. Cevc and D. Marsh, 1983, Calorimetric studies of the gel-fluid (L^-La) and lamellar-inverted hexagonal (La-Hn) phase transitions in dialkyl- and diacylphosphatidylethanolamines. Biochemistry 22, 1280-1289. 149. Barton, P.G. and F.D. Gunstone, 1975, Hydrocarbon chain packing and molecular motion in phospholipid bilayers formed from unsaturated lecithins, J. Biol. Chem. 250, 4470-4476. 150. Cevc, G., 1991, How membrane chain-melting phase transition temperature is affected by the lipid chain asymmetry and degree of unsaturation: An effective chain-length model. Biochemistry 30, 7186-7193. 151. Mattai, J., P.K. Sripada and G.G. Shipley, 1987, Mixed chain phosphatidylcholine bilayers: Structure and properties. Biochemistry 26, 3287-3297. 152. Wang, Z., H. Lin and C. Huang, 1990, Differential scanning calorimetry study of a homologous series of fully hydrated saturated mixed chain C(X):C(X+6) phosphatidylcholines. Biochemistry 29, 7072-7076. 153. Lin, H., Z. Wang and C. Huang, 1991, The influence of acyl chain-length asymmetry on the phase transition parameters of phosphatidylcholine dispersions, Biochim. Biophys. Acta 1067, 17-28, 154. Laggner, P, K. Lohner, G. Degovics, K. Miiller and A. Schuster, 1987, Structure and thermodynamics of the dihexadecylphosphatidylcholine-water system, Chem. Phys. Lipids 44, 31-60. 155. Lohner, K., A. Schuster, G. Degovics, K. Miiller and P. Laggner, 1987, Thermal phase behaviour and structure of hydrated mixtures between dipalmitoyl- and dihexadecylphosphatidylcholines, Chem. Phys. Lipids 44, 61-70. 156. Kim, J.T., J. Mattai and G.G. Shipley, 1987, Gel phase polymorphism in ether-linked dihexadecylphosphatidylcholine bilayers. Biochemistry 26, 6592-6598.

156

JM Seddon and R.H. Templer

157. Kim, J.T., J. Mattai and G.G. Shipley, 1987, Bilayer interactions of ether- and ester-linked phospholipids: Dihexadecyl- and dipalmitoylphosphatidylcholines. Biochemistry 26, 6599-6603. 158. Haas, N.S., P.K. Sripada and G.G. Shipley, 1990, Effect of chain linkage on the structure of phosphatidylcholine bilayers, Biophys. J. 57, 117-124. 159. Cevc, G., 1987, How membrane chain melting properties are regulated by the polar surface of the lipid bilayer, Biochemistry 26, 6305-6310. 160. Boggs, J.M., 1987, Lipid intermolecular hydrogen bonding: Influence on structural organization and membrane function, Biochim. Biophys. Acta 906, 353-404. 161. Janiak, M.J., D.M. Small and G.G. Shipley, 1979, Temperature and compositional dependence of the structure of hydrated dimyristoyl lecithin, J. Biol. Chem. 254, 6068-6078. 162. Kuttenreich, H., H.-J. Hinz, M. Inczedy-Marcsek, R. Koynova, B. Tenchov and P. Laggner, 1990, Polymorphism of synthetic l,2-0-dialkyl-3-0-/?-D-galactosyl-sn-glycerols of different alkyl chain lengths, Chem. Phys. Lipids 47, 245-260. 163. Sen, A., S.-W. Hui, D.A. Mannock, R.N.A.H. Lewis and R.N. McEIhaney, 1990, The physical properties of glycosyl diacylglycerols, 2. X-ray diffraction studies of a homologous series of l,2-di-0-acyl-3-0-(a-D-glucopyranosyI>sn-gIycerols, Biochemistry 29, 7799-7804. 164. Hinz, H.-J., H. Kuttenreich, R. Meyer, M. Renner, R. FrOnd, R. Koynova, A.I. Boyanov and B.G. Tenchov, 1991, Stereochemistry and size of headgroups determine structure and phase behaviour of glycolipid membranes: Densitometric, calorimetric and X-ray studies, Biochemistry 30, 5125-5138. 165. Tenchov, B.G., A.I. Boyanov and R.D. Koynova, 1984, Lyotropic polymorphism of racemic dipalmitoylphosphatidylethanolamine. A differential scanning calorimetry study. Biochemistry 23, 3553-3558. 166. Rand, R.P., N. Fuller, V.A. Parscgian and D.C. Rau, 1988, Variation in hydration forces between neutral phospholipid bilayers: Evidence for hydration attraction, Biochemistry 27, 7711-7722. 167. Tate, M.W. and S.M. Gniner, 1987, Lipid polymorphism of mixtures of dioleoylphosphatidylethanolamine and saturated and monosaturated phosphatidylcholines of various chain lengths, Biochemistry 26, 231-236. 168. Boni, L.T. and S.W. Hui, 1983, Polymorphic phase behaviour of dilinoleoylphosphatidylethanolamine and palmitoyloleoylphosphatidylcholine mixtures: Structural changes between hexagonal, cubic and bilayer phases, Biochim. Biophys. Acta 731, 177-185. 169. Eriksson, P.-O., G. Lindblom and G. Arvidson, 1985, NMR studies of 1- palmitoyllysophosphatidylcholine in a cubic liquid crystal with a novel stiiicture, J. Phys. Chem. 89, 1050-1053. 170. Cevc, G., 1990, Membrane electrostatics, Biochim. Biophys. Acta 1031, 311-382. 171. Fernandez, M.S. and P. Fromhertz, 1977, Lipoid pH indicators as probes of electrical potential and polarity in micelles, J. Phys. Chem. 81, 1755-1761. 172. Mabrey, S. and J. Sturtevant, 1977, Interaction of saturated fatty acids into phosphatidylcholine bilayers, Biochim. Biophys. Acta 486, 444-450. 173. Heimburg, T., N.J.P Ryba, U. WUrz and D. Marsh, 1990, Phase transition from a gel to a fluid phase of cubic synmietry in dimyristoylphosphatidylcholine / myristic acid (1:2, mol/mol) bilayers, Biochim. Biophys. Acta 1025, 77-81. 174. Hope, M.J. and PR. Cullis, 1981, The role of non-bilayer lipid structures in the fusion of human erythrocytes induced by lipid fiisogens, Biochim. Biophys, Acta 640, 82-90. 175. Tilcock, C.P.S. and D. Fisher, 1982, Interactions of glycerol monoolcatc and dimcthylsulphoxide with phospholipids: A differential scanning calorimetry and ^ip-NMR study, Biochim. Biophys. Acta 685, 340-346. 176. Gutman, H., G. Arvidson, K. Fontell and G. Lindblom, 1984, ^ip and ^H NMR studies of phase equilibria in the three component system: Monoolein-dioleoylphosphatidylcholine-dioleoylphosphatidylethanolamine, in: Surfactants in Solution, Vol. 1, eds K.L. Mittal and B. Lindman (Plenum, New York) pp. 143-152. 177. Nilsson, A., A. Holmgren and G. Lindblom, 1991, Fourier-transform infrared spectroscopy study of dioleoylphosphatidylcholine and monooleoylglycerol in lamellar and cubic phases. Biochemistry 30, 2126-2133.

Polymorphism of lipid-water systems

157

178. Caffrey, M., 1987, Kinetics and mechanism of transitions involving the lamellar, cubic, inverted hexagonal, and fluid isotropic phases of hydrated monoglycerides monitored by time-resolved X-ray diffraction. Biochemistry 26, 6349-6363. 179. Dawson, R.M.C., R.F. Irvine, J. Bray and P.J. Quinn, 1984, Long-chain unsaturated diacylglycerols cause a perturbation in the structure of phospholipid bilayers rendering them susceptible to phospholipase attack, Biochem. Biophys. Res. Commun. 125, 836-842. 180. Das, S. and R.R Rand, 1984, Diacylglycerol causes major structural transitions in phospholipid bilayer membranes, Biochem. Biophys. Res. Commun. 124, 491-496. 181. Das, S. and R.R Rand, 1986, Modification by diacylglycerol of the structure and interaction of various phospholipid bilayer membranes. Biochemistry 25, 2882-2889. 182. Epand, R.M., 1985, Diacylglycerols, lysolecithin, or hydrocarbons markedly alter the bilayer to hexagonal phase transition temperature of phosphatidylethanolamines. Biochemistry 24, 70927095. 183. Epand, R.M. and R. Bottega, 1988, Determination of the phase-behaviour of phosphatidylethanolamine admixed with other lipids and the effects of calcium chloride - implications for protein kinase-C regulation, Biochim. Biophys. Acta 944, 144-154. 184. Siegel, D.R, J. Banschbach and RL. Yeagle, 1989, Stabilization of Hn phases by low levels of diglycerides and alkanes: Calorimetric, and X-ray diffraction study, Biochemistry 28, 5010-5019. 185. Siegel, D.R, J. Banschbach, D. Alford, H. Ellens, L.J. Lis, RJ. Quinn, RL. Yeagle and J. Bentz, 1989, Physiological levels of diacylglycerols in phospholipid membranes induce membrane fusion and stabilize inverted phases. Biochemistry 28, 3703-3709. 186. Noordam, RC, C.J.A. van Echteld, B. de Kruijff and A. Verkleij, 1980, Barrier characteristics of membrane model systems containing unsaturated phosphatidylethanolamines, Chem. Rhys. Lipids 27, 221-232. 187. Dekker, C.J., W.S.M. van Kessel, J.RG. Klom, J. Pieters and B. de Kruijff, 1983, Synthesis and polymorphic phase behaviour of polyunsaturated phosphatidylcholines and phosphatidylethanolamines, Chem. Rhys. Lipids 33, 93-106. 188. Hornby, A.R and RR. Cullis, 1981, Influence of local and neutral anaesthetics on the polymorphic phase preferences of egg yolk phosphatidylethanolamine, Biochim. Biophys. Acta 647, 285-292. 189. Kirk, G.L. and S.M. Gruner, 1985, Lyotropic effects of alkanes and headgroup composition on the L(alpha)-Hn lipid liquid crystal phase transition: Hydrocarbon packing versus intrinsic curvature, J. Rhys. France 46, 761-769. 190. Sjolund, M., G. Lindblom, L. Rilfors and G. Arvidson, 1987, Hydrophobic molecules in lecithinwater systems, 1. Formation of reversed hexagonal phases at high and low water contents, Biophys. J. 52, 145-153. 191. Sj5lund, M., L. Rilfors and G. Lindblom, 1989, Reversed hexagonal phase formation in lecithinalkane-water systems with different acyl chain unsaturation and alkane length, Biochemistry 28, 1323-1329. 192. Lindblom, G., L. Rilfors, I. Brentel, R-0. Eriksson, G. Wikander, G. Arvidson and A. Wieslander, 1987, Effect of hydrophobic, amphiphilic and peptide molecules on phase structure and dynamics of membrane lipids, Chem. Scr. 27B, 221-227. 193. Lindblom, G., M. SjOlund and L. Rilfors, 1988, Effect of n-alkanes and peptides on the phase equilibria in phosphatidylcholine-water systems, Liquid Crystals 3, 783-790. 194. Gruner, S.M., M.W. Tate, G.L. Kirk, RT.C. So, D.C. Turner, D.T Keane, C.RS. Tilcock and RR. Cullis, 1988, X-ray diffraction study of the polymorphic behaviour of N-methylated dioleoylphosphatidylethanolamine. Biochemistry 27, 2853-2866. 195. Rand, R.R, N. Fuller, S.M. Gruner and V.A. Parsegian, 1990, Membrane curvature, lipid segregation, and structural transitions for phospholipids under dual-solvent stress. Biochemistry 29, • 76-87. 196. Lohner, K., 1991, Effects of small organic molecules on phospholipid phase transitions, Chem. Rhys. Lipids 57, 341-362. 197. Tenchov, B., 1991, On the reversibility of the phase transitions in lipid-water systems, Chem. Phys. Lipids 57, 165-177.

158

JM. Seddon and R.H. Templer

198. Chen, S.C., J.M. Stuitevant and B.J. Gaffney, 1980, Scanning calorimetric evidence for a third phase transition in phosphatidylcholine bilayers, Proc. Nat. Acad. Sci. USA 77, 5060-5063. 199. Ruocco, M.J. and G.G. Shipley, 1982, Characterization of the sub-transition of hydrated dipalmitoylphosphatidylcholine, Biochim. Biophys. Acta 684, 59-66. 200. Finegold, L. and M.A. Singer, 1986, The metastability of saturated phosphatidylcholines depends on the acyl chain length, Biochim. Biophys. Acta 855, 417-420. 201. Lewis, R.N.A.H., N. Mak and R.N. McElhaney, 1987, A differential scanning calorimetric study of the thermotropic phase behaviour of model membranes composed of phosphatidylcholines containing linear saturated fatty acyl chains. Biochemistry 26, 6118-6126. 202. Yang, C.R, M.C. Wiener and J.F. Nagle, 1988, New phases of DPPC/water mixtures, Biochim. Biophys. Acta 945, 101-104. 203. Chang, H. and R.M. Epand, 1983, The existence of a highly ordered phase in fully hydrated dilauroylphosphatidylethanolamine, Biochim. Biophys. Acta 728, 319-324. 204. Mantsch, H.H., S.C. Hsi, K.W. Butler and D.G. Cameron, 1983, Studies on the thermotropic behaviour of aqueous phosphatidylethanolamines, Biochim. Biophys. Acta 728, 325-330. 205. Chowdry, B.Z., G. Lipka, A.W. Dalziel and J.M. Sturtevant, 1984, Multicomponent phase transitions of diacyl phosphatidylethanolamines, Biophys. J. 45, 901-904. 206. Wilkinson, D.A. and J.E Nagle, 1984, Metastability in the phase behaviour of dimyristoylphosphatidylethanolamine bilayers, Biochemistry 23, 1538-1541. 207. Xu, H., F.A. Stephenson, H. Lin and C. Huang, 1988, Phase metastability and supercooled metastable state of diundecanoylphosphatidylethanolamine bilayers, Biochim. Biophys. Acta 943, 63-75. 208. Koynova, R. and H.-J. Hinz, 1990, Metastable behaviour of saturated phosphatidylethanolamines: A densitometric study, Chem. Phys. Lipids 54, 67-72. 209. Mason, J.T. and RA. Stephenson, 1990, Thermotropic properties of saturated mixed acyl phosphatidylethanolamines, Biochemistry 29, 590-598. 210. Mulakutla, S. and G.G. Shipley, 1984, Structure and thermotropic properties of phosphatidylethanolamine and its N-methyl derivatives. Biochemistry 23, 2514-2519. 211. Wilkinson, D.A. and T.J. Mcintosh, 1986, A subtransition in a phospholipid with a net charge, dipalmitoylphosphatidylglycerol, Biochemistry 25, 295-298. 212. Reed, R.A. and G.G. Shipley, 1987, Structure and metastability of N-lignocerylgalactosylsphingosine (cerebroside) bilayers, Biochim. Biophys. Acta 896, 153-164. 213. Mannock, D.A., R.N.A.H. Lewis, A. Sen and R.N. McElhaney, 1988, The physical properties of glycosyldiacylglycerosls. Calorimetric studies of a homologous series of l,2-di-0-di-3-0-(i9-Dglucopyranosyl)-sn-glycerols, Biochemistry 27, 6852-6859. 214. Tsong, T.Y., 1974, Kinetics of the crystalline to liquid-crystalline phase transition of dimyristoyl L-(alpha)-lecithin bilayers, Proc. Nat. Acad. Sci. USA 71, 2684-2688. 215. Strehlow, U. and E J^nig, 1981, Electrostatic interactions at charged lipid membranes. Kinetics of the electrostatically triggered phase transition, Biochim. Biophys. Acta 641, 301-310. 216. GrUnewald, B., 1982, On the phase transition kinetics of phospholipid bilayers. Relaxation experiments with detection of fluorescence anisotropy, Biochim. Biophys. Acta 687, 71-78. 217. Ranck, J.L., L. Letellier, E. Schechter, B. Krop, R Pemot and A. Tardieu, 1984, X-ray analysis of the kinetics of Escherichia coli lipid and membrane structural transitions, Biochemistry 23, 4955-4961. 218. Caffrey, M. and D.H. Bilderback, 1984, Kinetics of the main phase transition of hydrated lecithin monitored by real-time X-ray diffraction, Biophys. J. 45, 627-631. 219. Caffrey, M., 1985, Kinetics and mechanism of the lamellar gel - lamellar liquid-crystal and lamellar / inverted hexagonal phase transition in phosphatidylethanolamine: A real-time X-ray diffraction study using synchrotron radiation. Biochemistry 24, 4826-4844. 220. Laggner, R, K. Lohner and K. MOller, 1987, X-ray cinematography of phospholipid phase transformations with synchrotron radiation, Mol. Cryst. Liq. Cryst. 151, 373-388. 221. Caffrey, M., 1989, The study of lipid phase transition kinetics by time-resolved X-ray diffraction, Annu. Rev. Biophys. Biophys. Chem. 18, 159-186. 222. Gruner, S.M., 1987, Time-resolved X-ray diffraction of biological materials, Science 238, 305-312.

Polymorphism of lipid-water systems

159

223. Caffrey, M., R.L. Magin, B. Hummel and J. Zhang, 1990, Kinetics of the lamellar and hexagonal phase transitions in phosphatidylethanolamine. Time-resolved X-ray diffraction study using a microwave-induced temperature jump, Biophys. J. 58, 21-29. 224. Laggner, P. and M. Kriechbaum, 1991, Phospholipid phase transitions: Kinetics and structural mechanisms, Chem. Phys. Lipids 57, 121-145. 225. Tate, M.W., E. Shyamsunder, S.M. Gruner and K. D'Amico, 1992, Kinetics of the lamellar-inverse hexagonal phase transition determined by time-resolved X-ray diffraction, Biochemistry 31, 10811092. 226. Gulik, A., V. Luzzati, M. de Rosa and A. Gambacorta, 1985, Structure and polymorphism of bipolar isopranyl ether lipids from archaebacteria, J. Mol. Biol. 182, 131-149. 227. Shyamsunder, E , S.M. Gruner, M.W. Tate, D.C. Turner, P.T.C. So and C.RS. Tilcock, 1988, Observation of inverted cubic phase in hydrated dioleoylphosphatidylethanolamine membranes, Biochemistry 27, 2332-2336. 228. Siegel, D.P. and J. Banschbach, 1990, Lamellar/inverted cubic (La/Qn) phase transition in N-methylated dioleoylphosphatidylethanolamine. Biochemistry 29, 5975-5981. 229. de Kruijff, B., RR. Cullis, A.J, Verkleij, M.J. Hope, C.J.A. van Echteld, T.F. Taraschi, R van Hoogevest, J.A. Killian, A. Rietveld and A.T.M. van der Steen, 1985, Modulation of lipid polymorphism by lipid-protein interactions, in: Progress in Protein-Lipid Interactions, eds A. Watts and J.J.H.H.M de Pont (Elsevier, Amsterdam) pp. 89-142. 230. Gruner, S.M., 1985, Intrinsic curvature hypothesis for biomembrane lipid composition - a role for nonbilayer lipids, Proc. Nat. Acad. Sci. USA 82, 3665-3669. 231. Stier, A., S.A.E. Finch and B. BSsterling, 1978, Non-lamellar structure in rabbit liver microsomal membranes, FEBS Lett. 91, 109-112. 232. de Kruijff, B., A.M.H.P. van den Besselaar, PR. Cullis, H. van den Bosch and L.L.M. van Deenen, 1978, Evidence for isotropic motion of phospholipids in liver microsomal membranes, Biochim. Biophys. Acta 514, 1-8. 233. de Kruijff, B., A. Rietveld and RR. Cullis, 1980, ^^P-NMR studies on membrane phospholipids in microscomes, rat liver slices and intact perfused rat liver, Biochim. Biophys. Acta 600, 343-357. 234. Green, D.E., 1972, Membrane proteins: A perspective, Ann. NY Acad. Sci. 95, 150-172. 235. Cullis, RR., B. de Kruijff, M.J. Hope, R. Nayar, A. Rietveld and A.J. Verkleij, 1980, Structural properties of phospholipids in the rat liver inner mitochondrial membrane, Biochim. Biophys. Acta 600, 625-635. 236. Kachar, B. and T.S. Reese, 1982, Evidence for the lipidic nature of tight junction strands, Nature 296, 464-466. 237. Pinto da Silva, R and B. Kachar, 1982, On tight-junction structure. Cell 28, 441-450. 238. Verkleij, A.J., 1980, The nature of the intramembrane particle, Proc. Electr. Microsc. Soc. Am. 38, 688-691. 239. Meyer, H.W., W. Richter and J. Gumpert, 1990, Periodically curved bilayer structures observed in hyphal cells or stable L-form cells of a Streptomyces strain, and in liposomes formed by the extracted lipids, Biochim. Biophys. Acta 1026, 171-178. 240. Donnay, G. and D.L. Pawson, 1969, X-ray diffraction studies of echinoderm plates, Science 166, 1147-1150. 241. Nissen, H.-U., 1969, Crystal orientation and plate structure in echinoid skeletal units. Science 166, 1150-1152. 242. Corless, J.M. and M.J. Costello, 1981, Paracrystalline inclusions associated with the disk membranes of frog retinal rod outer segments, Exp. Eye Res. 32, 217-228. 243. Luzzati, V., A. Gulik, M. de Rosa and A. Gambacorta, 1987, Lipids from Sulfolobus solfactaricus, life at high temperature and the structure of membranes, Chem. Scr. B 27, 211-219. 244. Gunning, B.E.S., 1965, The greening process in plastids, I. The structure of the prolamellar body, Protoplasma 60, 111-130. 245. Simpson, D.J., 1978, Freeze-fracture studies on barley plastid membranes, I. Wild-type etioplast, Carlsberg Res. Commun. 43, 145-170. 246. Israelachvili, J.N. and J. Wolfe, 1980, The membrane geometry of the prolamellar body, Protoplasma 100, 315-321.

160

JM. Seddon and R.H. Tempter

247. Murakami, S., N. Yamada, M. Nagano and M. Osumi, 1985, Three-dimensional structure of the prolamellar body in squash etioplasts, Protoplasma 128, 147-156. 248. BorgstrOm, B., 1985, The micellar hypothesis of fat digestion: must it be revisited?, Scand. J. Gastroenterol. 20, 389-394. 249. Patton, J.S. and M.C. Carey, 1979, Watching fat digestion, Science 204, 145-148. 250. Rigler, M.W. and J.S. Patton, 1983, The production of liquid crystalline product phases by pancreatic lipase in the absence of bile salts, Biochim. Biophys. Acta 751, 444-454. 251. Rigler, M.W., R.E. Honkanen and J.S. Patton, 1986, Visualization by freeze fracture, in vitro and in vivo, of the products of fat digestion, J. Lipid Res. 27, 836-857. 252. Rand, R.P., 1981, Interacting phospholipid bilaycrs: measured forces and induced structural changes, Annu. Rev. Biophys. Bioeng. 10, 277-314. 253. Prestegard, J.H. and M.P. O'Brien, 1987, Membrane and vesicle fusion, Annu. Rev. Phys. Chem. 38,383-411. 254. Bentz, J. and H. Ellens, 1988, Membrane fusion - kinetics and mechanisms. Coll. Surf. 30, 65-112. 255. Verkleij, A.J., 1984, Lipidic intramembranous particles, Biochim. Biophys. Acta 779, 43-63. 256. Nishizuka, Y., 1984, The role of protein kinase-C in cell-surface signal transduction and tumour promotion. Nature 308, 693-698. 257. Berridge, M.J., 1987, Inositol triphosphate and diacylglycerol: Two interacting second messengers, Annu. Rev. Biochem. 56, 159-193. 258. Burger, K.N.J., J.-L. Nieva, A. Alonso and A.J. Verkleij, 1991, Phospholipase C activity-induced fusion of pure lipid model membranes. A freeze fracture study, Biochim. Biophys. Acta 1068, 249-253. 259. Ortiz, A., J. Villalain and J.C. G6mez-Femtodez, 1988, Interaction of diacylglycerols with phosphatidylchioline vesicles as studied by differential scanning calorimetry and fluorescence probe measurements, Biochemistry 27, 9030-9036. 260. Sinesky, M., 1974, Homeoviscous adaptation - a homeostatic process that regulates the viscosity of membrane lipids in Escherichia coli, Proc. Nat. Acad. Sci. USA 71, 522-525. 261. Cullen, J., M.C. Phillips and G.G. Shipley, 1971, The effects of temperature on the composition and physical properties of the lipids of Pseudomonasfluorescens,Biochem. J. 125, 733-742. 262. Lindblom, G., I. Brcntel, M. Sjelund, G. Wikandcr and A. Wieslander, 1986, Phase equilibria of membrane lipids from Acholeplasma laidlawii: Importance of a single lipid forming nonlamellar phases. Biochemistry 25, 7502-7510. 263. Wieslander, A., A. Christiansson, L. Rilfors and G. Lindblom, 1980, Lipid bilayer stability in membranes. Regulation of lipid composition in Acholeplasma laidlawii as governed by molecular shape. Biochemistry 19, 3650-3655. 264. Goldfine, H., N.C. Johnston, J. Mattai and G.G. Shipley, 1987, Regulation of bilayer stability in Clostridium butyricum: Studies on the polymorphic phase behaviour of the ether lipids, Biochemistry 26, 2814-2822. 265. Tournois, H. and B. de Kruijff, 1991, Polymorphic phospholipid phase transitions as tools to understand peptide-lipid interactions, Chem. Phys. Lipids 57, 327-340. 266. Sadoc, J.F. and J. Charvolin, 1989, Infinite periodic minimal surfaces and their crystallography in the hyperbolic plane. Acta Crystallogr. A45, 10-20.