J . Phys. Chem. 1989,93, 7304-7314
7304
Conclusions The weighted harmonic mean approach may be useful as a simple guideline in understanding and developing high- T, superconductors. Each atom has its unique net ability (eq 1) to La(Mno,5Ruo,5)03 5.10 L ~ ( C O ~ , ~ I ~ ~5.33 ,~)O~ exchange electrons with other different atoms surrounding it. La(N&Tio,5)03 5.20 L ~ ( M ~ o . s I ~ o ,5.24 ~O~ Thus, the complex structure for a high-T, oxide must be due to La(Nl0.5Rbd03 5.20 LaTiO, 5.15 the necessarily delicate balance of the atomic electronegativities La(Mg0,5Nb0,5)03 5.07 LaVO, 4.99 (eq 2) in order to create a potential field that is optimum for the " Reference 18. superconduction to occur. Studies of chemical bonds following the concept of electronegativity equilibration implies the imporfor the A15 alloys the xWHvalues are concentrated in the region tance of interactions through electrostatic force, a viewpoint shown of lower electronegativity. On the other hand, the nonsuperconductive oxides of perovskite to be valid for the case of YBa2Cu307.'9 structure (Table 111) have xWHvalues that are outside the critical It should be cautioned however that this type of argument has range for the high-T, oxides. This eliminates an anomaly found its limitations. Thus, the subtle difference in superconductive before3 that the average electronegativity of La(Mgo,5Nbo,5)03 properties of ordered or disordered structure (e.g., SnTa3) and falls into the same range for the high-T, oxides according to a normal or odd stoichiometry (e.g., the cases of Nb-Sn and Ge-Nb particular choice of scale, that is, xav= C ~ ~ , X J V ~ / C where ~ , ~ N ~ , systems) are not reflected in the values of xWH.Also, at least in one case for PbSb, the value of xWHfalls into the critical range xi is the Pauling electronegativity and Ni is the number of atoms of a particular species. There seems to be a roughly linear relation for high-T, oxides. Therefore, the method is not a sweeping generality. However, it seems to qualify as a convenient but between the electronegativities of Pauling and Mulliken so that effective way to eliminate or predict possible new superconductors. 4.7 eV for Tl2Ba2Ca2Cu3Ol0 corresponds to a value between 1.6 and 2.2 (median 1.9) on Pauling's scale which includes the range expected for individual elements2 This is in contrary to the results of Asokamani and M a n j ~ l a . ~ (19) Sawyer, D. T. J . Phys. Chem. 1988, 92, 8.
TABLE III: Weigbted Harmonic Mean Electronegativities of Nonsuwrconductive Oxides" oxide XWHI eV oxide XWH, eV
FEATURE ARTICLE Cubic Llpid-Water Phases: Structures and Biomembrane Aspects K. Larsson Department of Food Technology, Chemical Center, University of Lund, Box 124, S-221 00 Lund, Sweden (Received: February 15, 1989)
Recent studies of cubic lipid-water phases have shown that there are three basic structure alternatives, each consisting of an infinite periodically curved lipid bilayer separating two continuous water regions. The geometric factors that determine these structures are discussed. The biological relevance of cubic phases is related to the possibility of two-dimensional analogues of these structures, which are consistent with earlier observationsof so-called nonbilayer conformations in membranes. Evidence is given for phase transitions in membranes involving such intrinsically curved bilayers.
Introduction Cubic lipid-water phases form remarkable structures with perfect long-range three-dimensional periodicity, although the molecules exhibit a dynamical disorder at atomic distances like that in liquids. Monoolein is an example of a lipid forming such a phase, which can contain up to 40% (w/w) of water. It is transparent, very viscous, and can coexist in equilibrium with excess of water (with lipid monomer concentration as low as about 10" M). Due to the short-range disorder, these phases are called liquid crystals, although they really are plastic crystals. A review showing the occurrence of cubic phases in lipid systems of biological interest was recently reported.' Parts of the work in the determination of these most complex of the various lipid-water structures have been done in our laboratory. The general features of the molecular organization of ( 1 ) Lindblom, G.; Rilfors, L. Biochim. Biophys. Acta, in press.
0022-3654/89/2093-7304$01.50/0
cubic lipid-water phases will be reviewed below. In connection with the structural analysis of cubic phases it was realized that two-dimensional analogues to these phases provide a theoretical possibility for bilayer polymorphism of cell membranes. So-called nonbilayer conformations, which frequently have been discussed in the literature, are consistent with such periodic structures. This earlier nonbilayer description, however, is not adequate, as the structure still is a continuous bilayer, although intrinsically curved. Experimental data providing evidence for phase transitions in membranes involving such periodically curved bilayers will be given. Examples from ultrastructural studies of cell organelles and membranes will also be presented where this periodic structure clearly exists. Cubic Lipid-Water Phases This part will focus on the structure of cubic phases. Most of the results discussed below concern one particular type of lipid, fatty acid monoglycerides, which exhibit the three basic types of 0 1989 American Chemical Society
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The Journal of Physical Chemistry, Vol. 93, No. 21, 1989 7305
Figure 2. Lipid bilayer structure in lipid-water systems. The planar bilayer of the L, phase (to the left), structure unit of the HI,phase (in the middle), and a unit of a periodically curved cubic phase (to the right).
The two-dimensional lattice of water cylinders forming the H,phase is also indicated below the corresponding structure unit. 100
Figure 1. Crystal structure of the 2-monoglyceride of lauric acid.3
cubic structures. The results are relevant for all cubic phases of lipids. It should be pointed out, however, that a special type of cubic phase has been observed in a water-soluble type of lipid, lysophosphatidylcholines. This type of structure, consisting of a cubic packing of micellar aggregates in water, will not be considered further in this paper. I.1, General Behavior of Lipid-Water Systems: Relation between Molecular Shape and Structure. The unique property of lipid molecules from a physical/structural point of view is their association behavior in an aqueous environment. Thus, highly organized structures are formed possessing long-range order, although they are highly disordered on atomic distances, as mentioned above. Furthermore, such different liquid-crystalline phases exist for a particular lipid (polymorphism). The pioneering work on the nature of these phases was reported during the 1960s by Luzzati and co-workers (cf. ref 2). Some general features will be summarized below. An illustrative starting point for these discussions is the crystal structure. The general structure of lipids in the solid state is a stack of planar molecular bilayers. The hydrocarbon chains are close-packed in these bilayers, and the polar heads form the outer surfaces. As monoglyceride cubic phases will be treated in detail, this lipid is used to illustrate the solid-state packing of lipid^.^ Figure 1 shows the crystal structure of the 2-isomer, which is formed at lipolysis of fats and oils. The hydrocarbon chains have a high angle of tilt (44O), and the molecular cross section (26 A2) is almost as high as in the lamellar liquid-crystalline state. It is therefore not surprising that such a state is passed before melting, when these crystals are heated. The hydrogen-bond system in the sheets formed by the polar head groups is strong compared to the weak van der Waals interaction between the hydrocarbon chains; thus, the chains can "melt" although the polar groups still are associated into sheets. This liquid-crystalline phase, with long-range order in one dimension, is called L,. It is more common, however, that it is formed in the presence of water. Above the actual hydrocarbon chain melting temperature, water penetrates the polar region, and an La phase is formed, with water layers alternating with lipid bilayers (see Figure 2). There are also two other types of structures in lipid-water systems, as indicated in Figure 2, both based on the lipid bilayer. is best known. It consists of infinite water The hexagonal phase Hi, rods arranged in a two-dimensional lattice and separated by lipid bilayers. The structure unit can be described as intersecting bilayer units (Figure 2). The cubic structures, discussed in detail below, (2) Luzzati, V. In Biological Membranes; Chapman, R., Ed.; Academic Press: New York, 1968; p 71. ( 3 ) Larsson, K. Ark. Kemi 1964, 23, 23. (4) Hyde, S.T.;Ericsson, B.; Andersson, S.; Larsson, K . Z . Kristallogr. 1984, 168, 213.
I U
f!
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*/e 1 w/w) water Figure 3. Binary phase diagram of the 1-monoolein-water system.'
consist of curved infinite bilayers. These three types of phases exhibit one-, two-, and three-dimensional periodicity, and they can be unambiguously identified by their X-ray diffraction patterns. Membrane lipids show at least one of these phases. The effect of water content and temperature on the existence of the different phases is defined by the corresponding lipid-water phase diagram. The phase diagram of the monoglyceride of oleic acid is illustrated in Figure 3. This diagram will be considered further in the paragraph on the cubic structure determination. With increased hydrocarbon chain disorder, obtained either by heating or by increasing the water content, there is a transition from the L, phase to cubic phases (C, and C,) and finally into the HIIphase. There is extensive literature on phase properties of lipids, providing general relations between molecular structure and structure of corresponding aqueous phases (cf. ref 1 and 2). The molecules in the L, phase have the same molecular cross section area at the polar head groups as at the methyl end groups. With increased chain disorder, which increases from the "anchoring" at the polar heads toward the end of the chains, there is an obvious tendency of chain divergence. This can also be expressed as a molecular wedge shape. Transition from the La phase into the HI,phase will allow such increased molecular wedge shape. As will be shown below, the cubic phases are intermediate in wedge shape. A quantitative theory relating the wedge shape of the lipid molecules to the phase, which is formed with water, has been introduced by Israelachvili et al.5 It is remarkable how well the phase behavior can be predicted from this simple description of the molecular geometry. 1.2. Periodic Minimal Surfaces. Before consideration of the cubic lipid-water phases it is iatural to discuss periodic minimal surfaces, as they have turned out to be an important tool in the ( 5 ) Israelachvili, J.; Mitchell, J.; Ninham, B. J . Chem. Soc., Faraday Trans. 2 1976, 72, 1525.
7306 The Journal of Physical Chemistry, Vol. 93. No. 21. I989
Larsson
V
Figure 4. Plastic model showing the structure unit of the P surface. These units are joined along the three dimensions at the openings.
understanding of the cubic phases. A review involving the mathematical description was recently reported: and only a short description needed for the following structural discussions will be presented here. Minimal surfaces are well-known to us all from soap films. More than a century ago Plateau' described the analogue solution to the problem of minimization of surface area within a fixed boundary by soap films. The mathematical condition is that the average curvature is zero everywhere. The curvature of a surface is defined by the principal curvatures, cIand c2 If we imagine a plane through the normal to the surface, and this plane is allowed to rotate, it is possible to find a largest and a smallest value of the curvature at the intersection between this plane and the surface. These values are c1and c2 The mrage curvature at the actual point H is equal to II2(c1 + cz). The Gaussian curvature K is equal to c1c2. A surface with H = 0 in all points is called minimal surface. It is as convex as concave in all points. The Gaussian curvature is SO everywhere. A minimal surface can also exhibit crystallographic periodicity; it is then called an infinite periodic minimal surface (IPMS). The simplest of this structure type was derived by Schwarz,* and it is therefore called SchwarzIs primitive surface or P surface. This IPMS structure unit (constructed as a plastic model according to the corresponding soap film) is shown in Figure 4. Repetition of this unit in the x , y, and z directions gives the continuous P surface, separating two continuous and congruent channel systems. A general property of straight lines on an IPMS is that they constitute 2-fold axes of symmetry. The lines forming a square indicated in Figure 4 are such 2-fold axes. We can regard the joints in the x, y , and z directions as catenoids, formed by a soap film spanning adjacent squares in each direction. If these sets of soap film catenoids are put together, they will form a continuous surface, the P surface. There is another simple surface called the diamond or D surface, as the channel systems on each side form a diamond lattice. The analogous formation of this surface from a lattice of 2-fold axes is indicated in Figure 5. Catenoids spanning triangular frames (twisted 180° in adjacent planes) are directed upward alternating with downward. These catenoid systems in the four ( 1 1 1 ) directions fuse into one continuous surface, the D surface. There are three fundamental cubic minimal surfaces, related as discussed below: the P surface, the D surface, and the gyroid or the G surface. This last surface was discovered by Shoen? The most remarkable property is that it contains no straight lines. This surface is illustrated in Figure 6. There are other IPMS, which (6) Andersson, S.; Hyde, S. T.; Larsson, K.; Lidin, S. Chem. Reo. 1988, 88. 221. (7) Plateau, J. A. F. Statique exNrimenra1 et thzorique des liquides sorimis aux seulesforces molZculaires;Gauthier-Villars.Trubner et cie; 1873, 2 vols. (8) Schwarz, H. A. Gesammelte Mathematische AbhandlungeK Springer: Berlin; 1890. (9) Shoen, A. H. NASA Tech. Rep. 1970, No. 05541.
v
-
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-
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Figure 5. The lattice formed by the 2-fold axes of the D surface (the ( 1 1 1 ) plane). Filled and open circles indicate the catenoids going upward and downward, respectively. An illustration of the corresponding halves of these catenoids is shown below. The whole D surface is obtained by repeating these planes, with adjacent planes twisted 180' in relation to one another.
are more complex, but at present not one of these seems to be relevant in lipids. There is convincing evidence on the existence of cubic lipidwater phases with IPMS structures, where the lipid bilayer is centered on the surface with water filling the channel systems on each side. All three basic cubic IPMS structures discussed here, the P, D, and G surfaces, seem to exist. They are termed Cp,CD, and CG in the following text. There is an interesting relation between the Cp, CD, and CG structures, defined by the so-called Bonnet transformation. Thus, one of these surfaces can be continuously deformed into another, with constant average curvature and constant Gaussian curvature (isometric rela tionship). The general significance of IPMS has been demonstrated recently in simple crystal structures.6 von Schnering and NesperIO have shown that the equipotential or zero-potential surface in solids are identical with or close to an IPMS of the corresponding space group. The IPMS description of structures has also turned out to be useful in the analysis of various physical properties (cf. ref 6). Adsorption for example has been shown to be directly related to Gaussian curvature. It is interesting in this connection to consider the active sites in enzymes, which are regions of extreme Gaussian curvature.6 1.3. Structure Determination. Cubic phases are the most complex among the mesomorphic structures occurring in lipidwater systems. A general structure was proposed in 1968 by Luzzati and co-workers" consisting of interpenetrating rod networks. Thus, the polar head groups were suggested to form the rods and the hydrocarbon chains a continuous matrix. The structure determination was performed on X-ray data from an anhydrous lipid, strontium myristate.'* A different type of structure was later proposed according to studies of monoglyceride-water systems. Thus, lamellar lipid bilayer units forming a continuous lattice with water on each side were The main reason behind this proposal was the observation that the d(001) spacing of the L, phase persisted through the L, cubic phase transition coinciding with a cubic diffraction line. The NMR diffusion measurements showed that the structure was water- and lipid-continuous, and furthermore the lipid molecular diffusion was very similar to that of the L, phase. The proposed fused network of lipid bilayer disks was organized in agreement with the space group Im3m.
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(IO) von Schnering, H. G.; Nesper, R. Angew. Chem. 1987, 26, 1059. ( 1 1 ) Luzzati, V.; Tardieu, A.; Gulik-Kryzwicki, T.; Rivas, E.; RcissHusson, F. Narure 1968, 220,485. (12) Luzzati, V.; Spegt, P. A. Nature 1967, 215.701. ( 1 3) Lindblom, G.; Larsson, K.; Johansson, L.; Fontell, K.; Forstn, S. J . Am. Chem. Soc. 1979. 101. 5465.
Feature Article It was soon afterward realizedi4 that such a structure only needed a minor change from planar to curved disk units to be identical with the IPMS structure shown in Figure 4 (with a lipid monolayer on each side of the surface separating the water channel systems in the x , y, and z directions). Ironically enough, however, the cubic region of the monoolein-water phase diagram consisted of two cubic phases, the other two IPMS alternatives (CG and C,). As the two lattices are related, the Cp structure proposed is a “least common denominator”. The agreement between the two lattices, which provides strong evidence for the IPMS type of structures, is shown in Figure 7. This was revealed first by the work by Longley and M c I n t o ~ h , ’who ~ reported an X-ray analysis of the cubic phase at excess of water and also concluded that the structure was an IPMS. The lattice was indexed as a primitive one, with space group Pm3n, corresponding to the CD structure. In view of this result it was natural to reexamine other X-ray data of the whole cubic region,I6 which clearly showed that there were two cubic phases in this region, where first CG and then C, are formed on water swelling. The CG phase has space group I ~ 3 d The . ~ Cp phase also exists in monoolein-water systems with proteinsls and in protein-containing aqueous systems of another mainly unsaturated monoglyceride, that of sunflower oil.i9 An electron micrograph of this structure is shown in Figure 8. It should be pointed out that &riveni7 earlier had proposed that microemulsions and liquid-crystalline phases might form IPMS. It can be expected that a complete crystallographic determination of cubic lipid-water phases will not be possible, due to their high degree of disorder. Thus, X-ray diffraction lines are only observed a t spacings above about 25 A, which means that the resolution of any calculated electron density map can never be matched against the atomic distribution of the corresponding structure assumption. An impressive new approach to structural analysis was recently reported by Luzzati and ~ o - w o r k e rand s ~ ~their calculated electron density maps confirm the general principles of bicontinuous structures. The characterization of cubic phases should therefore also be based on evidence from other techniques, with the space group determination from the crystallographic data as the most important factor. It should be noticed, however, that even the space group determination can often not be done unambiguously, due to relatively few observed diffraction lines and the risk that absent reflections are accidental. Starting with the assumption that the lipid bilayer forms an IPMS, as concluded above, we should expect Cp, CD,or CG with space groups Im3m, Pm3n, and Ia3d, respectively. (Asdiscussed below there are also asymmetric bilayer versions of these.) The first problem is to determine whether the space group is primitive or body-centered. To be sure in this respect it is necessary to observe diffraction lines up to (321), which is the first reflection of a body-centered lattice with no correspondence in a primitive lattice indexing. In the case that a body-centered lattice can thus be identified strictly, it will be clear whether the space groups are Im3m or Ia3d from the absences. If only a few lines are observed, some guidance might be obtained from the intensity distribution in comparison with earlier reported cubic phases. An N M R technique has proved to be very useful and can show for example whether or not the structure is bicontinuous (cf. ref 1). Furthermore, freeze-fracture freeze-etching microscopy is a powerful technique, particularly if it is combined with X-ray diffraction (cf. ref 19).
(14) Larsson, K.; Fontell, K.; Krog, N. Chem. Phys. Lipids 1980, 27, 321. (15) Longley, W.; McIntosh, T. J. Nature 1983, 303, 612. (16) Larsson, K. Nature 1983, 304, 664. (17) Scriven, L. E. Nature 1976, 263, 123. (18) Buchheim, W.; Larsson, K. J . Colloid Interfuce Sci. 1987, 117, 582. (19) Gulik-Kryzwicki, T.; Aggerbeck, L. P.; Larsson, K. Surfuctnnts in Solution; Mittal, K. L., Lindman, B., Eds.;Plenum: New York, 1984; Vol. 1, p 237.
The Journal of Physical Chemistry, Vol. 93, No. 21, 1989 7307 In our calculations on the geometry of the cubic phases we have assumed that the polar groups are located on a parallel surface to the minimal surface. It is obvious that the effective length of the molecules must be related to the Gaussian curvature, and another alternative is that the polar groups are located at surfaces with H constant. A comparison has shown that there are very small differences in the structures discussed here. 1.4. Why Are Cubic Phases Cp, C,, or CG? Are the cubic IPMS structures discussed above just a topologically different alternative to the earlier rod description of these phases? The answer is no, as will be demonstrated below. Still the rod description represents an important step in the elucidation of the cubic structures. Perhaps the most important result by introducing the IPMS approach was that the infinite lipid bilayer nature of these phases was revealed:I4 the fact that a single bilayer with no self-intersection can separate two continuous water regions. If we consider models of cubic lipid water phases as IPMS with actual dimensions, the structure types Cp and CDlook like water globules separated by bilayers and fused in six or four lateral directions, respectively. Such water regions show no features of rod systems, and it makes no difference whether the polar heads are assumed to be located on constant average curvature surfaces or parallel surfaces in this respect. The difference between the IPMS structure and the corresponding rod description is most striking in the case of the CG structure. The most obvious feature of this structure, when viewing a plastic model as shown in Figure 9, is that the structure forms straight (infinite) helical tubes oriented parallel to the four (1 11) directions of the unit cell. In Figure 7 only the cylindrical core of the rod is seen. From the IPMS description it is also possible to understand which one of the three fundamental structures should be formed under the actual conditions. The most obvious requirement is that there must be space enough for the lipid bilayer. If we consider the Cpor the CDstructures, there are narrow necks in the surface at the points where the globular units join. The closest distance corresponds to the circular cross section of the catenoids between the actual nets. These circular necks of the Cp surface are seen in Figure 4. If we imagine a lipid bilayer centered on this surface, it is obvious that the thickness of the bilayer must be smaller than this circular cross section. This directly gives a lower limit of water content of the Cp phase. If we compare the CD phase, this limit is shifted to allow a higher bilayer content per unit volume. This explains why the Cp phase is not seen in the binary system monoolein-water (Figure 2), with a lipid content of more than 60% (w/w), whereas it exists when proteins are introduced and the bilayer takes up a smaller proportion of the unit volume (see next paragraph). The CG structure on the other side contains no circular necks, giving this compositional restriction. This explains why the CG structure can be formed at low water content even down to anhydrous lipids. Provided that the composition allows alternative structures, which phase is then formed? The critical factor on this level is the molecular wedge shape. The volume relations per unit surface of Cp, CD,and CG are 1.07, 1.02, and 1.00, respectively. If we also consider the constant-curvature surface or parallel surface, corresponding to the lipid bilayer centered on the surface, the molecular wedge shape can be calculated. In the case of the monoolein system (Figure 2) it is obvious that only the CG phase can form at the very low water content due to the constraints described above. Furthermore, it is clear that the swelling of the CG phase means a reduction in wedge shape, which is a change in the molecular shape contrary to the desired increase in disorder. Thus, the swelling builds up a strain, and the phase transition should thus be expected to release this strain. A molecular wedge shape factor can be defined as V/al, where Vis the molecular volume, a is the cross section area at the polar heads, and 1 is the molecular length. At the phase transition at 35% (w/w) of water, the calculated wedge shape of the CG phase of 1.27 is increased to 1.3 1 in the C, phase. The real molecular shape in the actual phase, as described by the wedge shape factor, is a compromise
7308 The Journal of Physical Chemistry, Vol. 93, No. 21, 1989 between the preferred shape based on molecular geometry only (corresponding to preferred curvature) and all the other forces acting on the molecules. It should be pointed out in this connection that a Bonnet transformation CG CDmeans a change in the surface content per unit volume. A perfect Bonnet transformation at heating or water swelling, keeping the Gaussian curvature constant, is therefore not possible. In our experimental4 results we also observed a corresponding “adjustment” of the lattice to account for the constant lipid-water composition. Thus, the a axis of the CD phase was shorter by a factor of 0.96 than that calculated from the CG axis, assuming a perfect Bonnet transformation. When considering the molecular wedge shape, it is of interest in relation to the biological problems discussed later to compare the cubic and hexagonal HI,phases in this binary system. The wedge shape factor is about 1.7 in the case of the HI, phase, whereas the cubic phases give wedge shape factors around 1.3. A comprehensive review of the cubic phases observed in various systems of surfactant molecules has recently been presented by Fontell.’* It should be pointed out that the cubic surfactant-water phases observed in a wide variety of systems exhibit the same structures according to X-ray diffraction and spectroscopy studies as the lipid systems discussed here. The mechanism of transition between L,, cubic, and HI,phases has been analyzed by Siegel (cf. ref 20) with special regard to its biological relevance. 1.5. Lipid-Protein- Water Phases. Cubic phases are also unique in their ability to accommodate proteins compared to other lipid-water phases. We have examined a wide range of globular proteins with molecular weights 5000-150000, all of which formed cubic phases, and a ternary lipid-protein-water phase diagram was also determined.21 The phase diagram of monoolein-water-lysozyme is shown in Figure 10. The protein incorporation also results in an increased water swelling. All three phases, Cp, CD,and CG, were foundI8 in these ternary lipid-protein-water systems. The protein molecules were shown to be located in the water channel systems. They were also found to keep their native structure, as proved by thermal analysis of the phase and measurements of enzymatic activity. Also, lipophilic protein molecules can be incorporated into the lipid bilayer of the cubic monoolein-water phase, as was demonstrated by a gliadin fraction. These composite cubic lipidprotein structures might be used as a method for matrix fixation of enzymes. Luzzati and co-workersZ3have recently reported a detailed structure analysis of cubic phases of different systems, one involving monoolein-cytochrome c-H20. Besides the earlier described IPMS structures, they also found space groups corresponding to asymmetry over the minimal surface. If the channel systems on each side are different, or if the lipid bilayer exhibits asymmetry, there are thus three other CD, Cp, or CG structures. 1.6. Dispersions of the Cubic Phase. If the cubic monoolein-water phase is shaken in a bile salt solution, a dispersion is formed with kinetic stability like a liposomal dispersion. In the polarizing microscope it is sometimes possible to see an outer birefringent layer with radial symmetry (showing an extinction cross like a liposome). The core, however, is isotropic. These dispersions are formed in the region of the ternary system, where the cubic phase exists in equilibrium with water and the L, phase. The dispersion is obviously due to a localization of the L, phase outside cubic particles. A possibility of linking a cubic core with an L, surface zone is indicated in Figure 11. The cubic phase can also be dispersed by strongly amphiphilic proteins. Caseins, for example, which also are very effective as emulsifiers, can disperse the cubic phase like simple surfactants,
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(20) Siegel, D. Chem. Phys. Lipids 1986, 42, 279. (21) Ericsson, B.; Larsson, K.; Fontell, K. Biochim. Biophys. Acta 1983,
729, 23. (22) Larsson, K.; Lindblom, G.J. Dispersion Sci. Technol. 1982, 3, 61. (23) Mariani. P.; Luzzati, V.; Delacroix, H. J. Mol. Biol. 1988, 204, 165.
Larsson such as bile salts. An electron microscopy study of such dispersion indicates that the periodicity of the IPMS bilayer is strongly increased in an outer zone containing proteins,18 making this zone highly hydrated and therefore easy to disperse.
Biological Aspects The first example in nature of a n IPMS was reported by Donnay and P a w ~ o n . *They ~ demonstrated that the echinoderm skeleton after removal of the organic matrix forms a P surface. In our first proposal of a minimal surface type of structure in monoglyceride-water systems,14 we also reported an IPMS structure of the prolamellar body, as described in Gunning’s beautiful ultrastructural analysis.25 The prolamellar body is a depot form of thylakoid membranes, and in the growing of cereal seedlings, for example, the prolamellar bodies develop into chloroplasts. In this first report of IPMS in a living system we also pointed out that the space group of such a P surface structure had to be Im3m, due to the non-Euclidean mirror symmetry provided by the surface. Patton and Careyz6demonstrated that the digestion of fat in globules in an in vitro system corresponding to the intestine results in the formation of a viscous isotropic phase on the surface. As monoglycerides and fatty acids are the enzymatic degradation products, it was natural to assume that they observed the cubic phase. We therefore analyzed a ternary system corresponding to the digestion of fat?’ and it was found that the cubic phase is dominating in such a system. An additional piece of information, which is interesting in this connection, is the dispersion of cubic phases discussed above. If we consider the requirement of water transport out from the site of hydrolysis and localization of the enzyme at the oil interface, it is obvious that the minimal surface type of structure described exhibits ideal properties. As discussed above, proteins can be incorporated into the water channel systems of the cubic phase. Thus, lipase can move through the cubic phase to the oil phase. Furthermore, water molecules can be removed from the oil/water interface, and the water activity can be kept under control. Aggregates of the cubic phase may play a direct role in fat digestion and absorption even to the final stage with the possibility of fusing of cubic bilayer units with the intestinal membrane. In fact, Borgstrom, one of the pioneers behind the present view of fat absorption, has recently proposed a revised view on the absorption mechanismz8which is consistent with the phase properties discussed above. The most important biological significance of the IPMS type of bilayer structure, however, concerns the structure and function of cell membranes. This subject will be discussed below. First, a hypothetical phase transition mechanism will be described. Examples supporting IPMS conformations in membranes from an ultrastructural point of view will be given. The cell membrane phase transition model has been analyzed by introducing bilayer perturbations expected to shift the transition, as described in the last part of this paper. II.1. The LiD F+. CDPhase TransitionModel for Cooperative Membrane Phenomena. If we consider the network with catenoids up and down shown in Figure 4, it is obvious that an upper and a lower intersection of this IPMS parallel to the network gives a two-dimensional minimal surface with ringlike openings directed up and down. The lipid bilayer of a membrane might form such a structure provided that the ”holes” are filled by protein molecules. Based on this possibility, a phase transition model involving such periodical curvature was proposed to be involved in cooperative membrane functions.29 Later work indicated a general lipid composition control mechanism in membranes utilizing such a tran~ition.~~ (24) (25) (26) (27)
Donnay, G.;Pawson, D. L. Science 1969, 166, 1147. Gunning, B. E. S . Protoplasma 1965,60, 11 1. Patton, J. S.; Carey, M. C. Science 1979, 204, 145. LindstrBm, M.; Ljusberg-Wahren, H.; Larsson, K.; Borgstrom, B.
Lipids 1981, 16, 749. (28) BorgstrBm, B. Scand. J. Gasfroenterol. 1985, 20, 389. (29) Larsson, K.; Andersson, S. Acfa Chem. Scand. 1986, 840, 1.
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The Journal of Physical Chemistry, Vol. 93, No. 21, 1989 7309 IA)
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b
figure 6. A plastic model of the G surface? The two sides have different colors.
An illustration of this transition, which can be called LiD + C2D,is given in Figure 12. Before we go into experimental observations, some of the obvious features of this type of transition will be summarized. The general driving force behind the unique' polymorphism of lipid bilayers in three dimensions (changes in the average molecular wedge shape) can also operate in a membrane bilayer by such a transition, and in fact only in this way provided that there always is an infinite bilayer. No model of a "nonbilayer" conformation of membranes has been presented which is related to known three-dimensional lipid structures. The lipid-protein interaction in the bilayer means that the phase transition into an "excitedn intrinsically curved conformation might be induced by a protein conformational change as well as by changes in lipid molecular wedge shape. A direct consequence of this is the possibility of controlling lipid composition by an on/off switch on the enzymes responsible for lipid modifications. Methyl transferase enzymes, for example, converting phosphatidylethanolamines (favoring C2Dconformations) into phosphatidylcholines (favoring LiD), might be activated in this way. The main evidence for the proposed control of the lipid composition in membranes monitored by a phase transition (LiD e CZD)switch is the extensive literature on the effect of environmental factors, like temperature, pressure,32and lipophilic agent^.^^^^^ An excellent review on further structural effects on membrane lipids, related to a La HIItransition, was recently reported by Cullis et al?5 It should be pointed out that their data also support an LzD C2Dtransition although they argue for the significance of the La HIItransition, as cubic structures are intermediate in relation to La/HII,and the present phase transition allows a bilayer conformation in membranes. Numerous membrane functions can be achieved by such a phase transition, for example cooperativity in receptor-ligand binding and fusion processes, or communication along the cell surface by phase transit ion waves. The driving force behind the transition can be related to &membranefluidity", which is an overall description of dynamic
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(30) Larsson, K. Chem. Phys. Lipids 1988, 69, 65. (31) Joseph, J. D.Prog. Lipid Res. 1979, 18, 1. (32) MacDonald, A. G. In Topics in Lipid Research; Klein, R. A., Schmitz, G. R., Eds.; Royal Society of Chemistry: London, 1986; p 319. (33) Boigegrain, R. A.; Fernandez, Y.; Massol, M.; Mitjavila, S. Chem. Phys. Lipids 1984, 35, 321. (34) Hilzemann, R. J.; Harris, R. A.; Loh, H. H. In Phospholipids in Cellular Regulation; Kuo, J. F., Ed.;CRC Press: Boca Raton, FL, 1986; Vol. 1, p 97. (35) Cullis, P. R.; Hope, M. J.; de Kruijff, B.; Verkleij, A. J.; Tilcock, C. P. S. In Phospholipids in Cellular Regulation; Kuo, J. F., Ed.; CRC Press: Boca Raton, FL, 1986; Vol. 1, p 1.
25
%I
35
40.
Figure 7. The first and dominating spacing versus water concentration in the binary system monoolein-water. In the case of DG and CDthe spacings are (21 I ) and (1 lo), respectively.
disorder traditionallyused in membrane research.36 An interesting aspect with regard to the tendency of molecular wedge shape and Cm mnformations is that the "viscosity" calculated for the surface of the bilayer is about 10 times higher than that in the center. Diffusion coefficients in membranes measured by different techniques are about 10-lo-lO-ll cm2/s in the case of proteins (in muscle cell or erythrocytes) and 10-8-10-9 cm2/s in the case of phospholipids (in vesicles and mitochondria). The hydrocarbon chain disorder of the lipid bilayer of a membrane is influenced by temperature and pressure. The general increase with temperature, involving a tendency for La cubic HIIphases, was discussed in the first paragraph. Less is known experimentally about pressure effects. X-ray studies3' and Raman spectroscopy studies38 have shown that pressure increases the trans/gauche ratio of the chains, which is reflected in an increased bilayer thickness and a decreased molecular cross section. In a review by M a c d ~ n a l don~ ~pressure effects on membranes and lipid bilayers, it is concluded that a pressure increase of 100 atm is equivalent to a temperature decrease in the range 2-8 "C, and from the volume and enthalpy changes associated with a bilayer La Hll.phasetransition, the Clausius-Clapeyron relationship gives a shrft in 2 "C in phase transition temperature per IO0 atm.39 Helfrich and co-workers have reported pioneering work on elastic energy of lipid bilayers (cf. ref 40), which also is relevant for membrane conformation. The bending elastic energy of a membrane per unit area is
-
-
where K is the binding rigidity and R is the elastic modulus of Gaussian curvature. Ro is Helfrich's "spontaneous radius of curvature" and R1,R2 are the principal radii of curvature. The equilibrium conformation corresponds to a minimum in elastic free energy. The ratio between K and iiwill determine the conformation, and three general solutions exist. One corresponds to one large vesicle and the other to numerous small ones. The third alternative is a "lattice of passages", which in the previous discussion corresponds to a three-dimensional minimal surface. It is interesting to note that the vesicle formation from bilayer systems of minimal surface types via a single bilayer can be explained by the elastic energy. (36) Lenaz, G.Biosci. Rep. 1987, 7, 823. (37) Stamatoft, J.; Guillon, D.; Powers, L.; Cladis, P. Biochem. Biophys. Res. Commun. 1978, 85, 724. (38) Yager, P.; Peticolas, W. J. Biophys. J . 1980, 31, 359. (39) Macdonald, A. G. Philos. Trans. R . SOC.London 1984, 8304, 47. (40) Habich, W.; Servuss, R. M.; Helfrich, W. 2. Nuturforsch. 1978,33, 1013.
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7310 The Journal of Physical Chemistry, Vol. 93, No. 21, 1989
Figure 8. Electron micrograph of a freeze-fractured and freeze-etched sample of sunflower oil monoglycerides/water/cytochrome c.I9 The sample was examined by X-ray diffraction before and after freezing. The relative displacement of the square lattice between adjacent fracture planes of half a period is consistent with a body-centered lattice and the Cp structure. Magnification: X 135000.
An elastic wave in a cell membrane, induced for example as a local conformational change by an electrical field, will propagate with a speed of about 200 m/s (speed of sound). There is an obvious difference between an L, phase and a cubic phase with regard to distribution of molecular disorder relevant here. In an L, phase all molecules have the same packing conditions and thus the same disorder constraints. In the cubic Cp CD,or CGstructures, however, there is a continuous variation in packing conditions, from flat points to the points with highest Gaussian curvature, and the disorder should be expected to increase with the Gaussian curvature. In a membrane bilayer the lipid molecules in an LiDconformation are statistically distributed, whereas a C2Dconformation should be expected to exhibit lateral segregation. Thus, the higher the Gaussian curvature, the higher the relative proportion of cis double bonds or other features related to increased disorder. Protein accommodation in the lipid bilayer is favored by disorder, and in a lipid bilayer with lateral segregation according to disorder, the proteins will be 'squeezed" into positions of highest disorder. Thus, there is a driving force for proteins toward the "holes" in the C2Dbilayer structure. Furthermore, there is a lateral packing pressure to make the protein region spanning the bilayer (or monolayer in the case of peripheral proteins) follow the average hydrocarbon chain direction. This would mean that ideally there would be proteins filling the "hole" of the CZDlipid bilayer and with a wedge shape as indicated in Figure 12. The actual proteins may not meet this requirement, and the ideal case in Figure 12 corresponds to the "conformational pressure" induced by the lipid bilayer. Such an anisotropy in lateral pressure on a protein provides a simple switch mechanism for enzymatic reactions, or it might close or open channels. It is also obvious that not all protein molecules need to fit the "holes". These other rotein molecules will possess lateral diffusion freedom as in the L: conformation. The fixed protein positions are kept exposed as long as the C2Dconformation exists. Finally, the lipid bilayer overall shape in a C2Dconformation can also vary, as shown in Figure 13. The lipid asymmetry may mean that there is only "holes" in one direction. Furthermore, we have only considered minimal surfaces, i.e., H = 0. The general curvature of membranes, however, means that H = constant > 0 should be a relevant alternative. In this connection it should be pointed out that variations in the average curvature are one way of the membrane to modulate its sensibility to osmotic pressure differences over the membrane (the Laplace-Young q u a tion).
E
In an earlier work the detailed geometry of the C2Dconformation has been analyzed?' based on a Cp(100) or CD(1 1 1) bilayer, involving variations in "hole" distances and amplitude of the curvature. II.2. Examples of Observed Membrane Periodicity Consistent with IPMS Structures. There are numerous reports in the literature on periodic structures of membranes according to electron microscopy studies. A three-dimensional periodic membrane structure has been observed to fill almost the whole glandular cells of the catfish dendritic organ.42 There are clear indications of an IPMS type of structure from the reported electron micrographs; the cytoplasmic tubular network shows a texture corresponding to the CG structure. In electron micrographs from studies of the inner membrane organization in mitochondria and chloroplasts periodic structures like those described above are often seen. A major difference between these two ATP-driven proton pump systems is the open and continuous structure of the mitochondrial cristae compared to the discontinuous stacks of closed thylakoid sacs. The cristae itself might thus behave as an IPMS structure of the bilayer, the periodicity providing an organization of the enzymes corresponding to the sequential reactions of the respiratory chain. The periodic structures of the chloroplasts have been observed at the interface (appressed regions) between the thylakoid disks in the granum43 and may have the role of stack organization. There are in fact differences in the monogalactosyldiglyceride/digalactosyldiglyceride (MGDG/DGDG) ratio between appressed and nonappressed regions.& DGDG forms an L, phase, whereas MGDG favors a cubic or H,t-ypeof structure.' Phase studies of chloroplast MGDG/DGDG lipid mixtures in water45have shown electron micrographs, which now can be interpreted as corresponding to cubic phases with IPMS structures. The examples above, as well as the pmlamellar body, represent three-dimensional membrane assemblies. It Seems quite obvious that the IPMS structure provides a mechanism of organizing such assemblies. Furthermore, there are no structural alternatives known from lipid-water phase studies which exhibit the observed (41) Hyde, S.; Anderson, S.; Larson, K. Z.Kristallogr. 1986,174,237. (42) van Lcnnep, E. W.; Lanzing, W. J. R. J . Ultrustruct. Res. 1961, 18, 333. (43) Miller, K. R. Sci. Am. 1979, 24, 102. (44) Murphy, D. J.; Woodrow, I. E. Biuchim. Biophys. Acta 1983,725, 104.
(45) Quinning, P. J.; Williams, W. P. Biuchim. Biophys. Acta 1983,737, 223.
Feature Article periodic textures. A next level of organized membrane systems is layers of double or multiple membranes forming cell surface zones, and also in such systems there are many examples of periodic IPMS-like arrangements. The milk fat globule is covered by a protein-rich (mainly xanthine oxidase) aqueous layer and then the fat globule membrane. The distance between the fat globule, with a polar lipid surface, and the real membrane, which originates from the plasma membrane of the secretory cell, is about 100-500 A. The interfacial zone where the fat globule adheres to the fat globule membrane is a periodic structure.a It shows the appearance of a CDstructure, and the periodicity is about 200 A. Another double membrane, which is periodically curved, has been observed in the intestinal wall of an insect.47 Different types of periodic structures forming envelopes of microorganisms have been reported. Gram-negative bacteria exhibit an inner and outer “double track”, each about 80 A thick. The inner one is the plasma membrane. There are many cases where the outer double track exhibits periodic structures like IPMS (see ref 48 and its references). These examples can be seen as IPMS curvature control of long-range organization, involving membranes. A common feature between the milk fat globule membrane and the double membrane of Gram-negative bacteria is the periodic structure in between. We can imagine the IPMS structure shown in Figure 5, consisting only of two adjacent networks: the upper with only catenoids downward fused with a bottom network with catenoids upward. The textures reported in the membrane systems described above are consistent with such a mechanism of membrane coupling. More interesting with regard to the proposed phase transition model is, however, the observations of periodicities in single plasma membranes. There are numerous reports on “orthogonal arrays of particles (OAP)” visible in freeze-fractured replices of plasma membranes from glial cells, skeletal muscle cells, and transport epithelia cells (ref 50 and its references). The appearance of this structure corresponds to the Cpstructure (see Fi ure 14). There are patches of this structure in a disordered (L,b) environment. OAP have been assumed to provide a control mechanism for membrane rigidity and stabilization of u n d u l a t i ~ n s . ~It~is obvious that the structure is due to a periodically curved bilayer, and the “particle” appearance is a consequence of the curvature. The periodic structure of Gram-negative bacteria was earlier identified with the lipoprotein fraction (the outer membrane) in studies of Spirilliu m serpens .49 Membranes of the Gram-positive bacterial strain Streptomyces hydroscopicus exhibit periodic membrane structures. These organized structures have been examined in viable L-form (protoplast) cells. The EM texture was interpreted as consisting of a cubic organization of vesicles at the cytoplasmic side of the membrane.s2 Also this structure, however, is in complete agreement with the IPMS type of structure described here. Freeze-fractured replices of plasma membranes of Paramecium tetraurelia show rectilinear arrays of particles, which are proposed to function as chemorecept~rs.~~ This structure is consistent with the Cp lipid bilayer structure described here. Finally, it should be pointed out that there are numerous reports on model systems of membrane lipids, which exhibit periodic structures with electron microscopy textures like the cubic phases, for example, “intramembrane particles” or “lipidic particles”. Lipidic particles were first observed in pure lipid water systems.” Extensive studies by Lindblom and co-workers’ have shown that (46) Buchheim, W. Natunvissenschafren 1982, 69, 505. (47) Lane, N. J.; Harrison, J. B. J . Cell Biol. 1979, 39, 353. (48) Watson, S.W.; Remsen, C. C. J . Ulrrastruct. Res. 1970, 33, 148. (49) Murray, R. G. E. Can. J. Microbiol. 1963, 9, 381. (50) Hirsch, M.; Gache, D.; Noske, W. Cell Tissue Res. 1988,252, 165. (51) Gotow, T. J . Neurocyrol. 1984, 13, 431. (52) Sternberg, B.; Gumpert, J.; Reinhardt, G.; Gawrisch, K. Biochim. Biophys. Acra 1987, 898. 223. (53) Preston, R. R.; Newman, T. M. J . CellSci. 1986, 83, 269. (54) Verkeig, A. J. Biochim. Biophys. Acra 1984, 779, 43.
The Journal of Physical Chemistry, Vol. 93, No. 21, 1989 7311 all lipid systems reported to give lipidic particles also form cubic phases. These aggregates have been related to membrane functions, and the occurrence of the cubic phase has probably direct relevance for membrane fusion.’ A lipid-protein system exhibiting intramembrane particles has also been reported. It was obtained by reconstitution of the major intrinsic protein of the eye lens fiber into liposomes. The structure reported corresponds to both Cp and CD, applying the IPMS d e s c r i p t i ~ n .Intramembrane ~~ particles have also been observed a t cell junctions (cf. ref 5 6 ) , and their structure was related to the HIIphase. The periodic texture observed, however, is consistent with an IPMS structure. 11.3. Neuronal Membranes and Perturbants: Anesthetic Effects. The bilayer of nerve cells consists of very specific lipids, and they are extremely sensitive for minor amounts of amphiphilic or lipophilic compounds. If the LiD C m phase transition would be involved in the propagation of the action potential, a shift in the transition in any direction should be expected to result in an anesthetic effect. It therefore seemed natural to try to analyze anesthetic agents with regard to effects on the L, F= cubic transition in a relevant lipid systems. General anesthetic agents like chloroform were observed to induce a phase transition L, which obviously corresponds to blocking of the sodium channels. If we consider the bilayer transition, as illustrated in Figure 12, it seems obvious that the curved conformation can induce a conformational change of an ion channel protein. The effects were also examined in a model system exhibiting both L, and cubic phases in excess of water, and the result is shown in Figure 15. At small amounts of the anesthetic agent, the L, phase cannot exist. The concentrations needed in order to influence the phase transition were also found to be correlated with the general anesthetic potency.57 It is generally accepted that general anesthetic agents influence the lipid bilayer order. The reversion of the anesthetic effect with pressure is also consistent with the phase transition effect; a pressure increase can give CZD LiD (see section 11.1). Local anesthetics seem to shift the phase transition in the opposite direction. Lidocain hydrochloride, for example, has no effect on a phospholipid liposomal dispersion (beside giving two L, phasess7). The cubic phase of monoolein, however, is transformed into the L, phase.58 Local anesthetic agents are considered to either perturb the lipid bilayer or exhibit a specific effect on the sodium channels (cf. ref 59). Many observations are consistent with the simple concept of a phase transition shift. Divalent ions, for example, should be expected to induce a higher average wedge shape of the lipid molecules (at cross-linking anionic phospholipids). It is therefore not surprising that they have antagonistic effects in relation to local anesthetic agents.60 Observations on the effect of anesthetic agents on enzyme systems controlling neuronal membrane compositions are also consistent with these phase transition shifts. Thus, inhibition of methyl transferase activity and effects of anesthetics on sialidase degradation of gangliosides61 can be related to an on/off switch of enzymatic activity provided by an LiD CZDtransition. It should be pointed out that the ion influx at the spike will induce a tendency toward an increased average wedge shape of the molecules, due to shielding of the lateral repulsion of phosphatidylserine molecules. The high compositional asymmetry does in fact indicate that the two monolayers strive toward different bilayer conformations. The outer membrane should thus tend to be in an L, conformation, whereas the inner one (with high
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(55) Dunia, I.; Marenti, S.;Rousselat, A.; Benetti, E. L. J . Cell Biol. 1987, 105, 1679. (56) Feltkamp, C. A.; van der Waerden, J. A. W. J . Cell Sci. 1983,63, 271. (57) Larsson, K. Langmuir 1988, 4, 215. (58) EngstrBm, S. Work in progress in our laboratory. (59) Escudero, B.; Gutidrrez-Merino. L. Biochim. Biophys. Acra 1987, 902, 374. (60) Seelig, A.; Allegrini, P. R.; Seelig, J. Biochim. Biophys. Acra 1988, 939, 267. (61) Scheel, G.; Acevedo, E.; Conzelmann, E.; Nehrkorn, H.; Sandoff, K. Eur. J . Biochem. 1982, 127, 245.
7312 The Journal of Physical Chemistry, Vol. 93, No. 21, 1989
Larsson
n /-
0
-
Figure 11. An aqueous dispersion of the cubic phase (“cubosomes”). A cross section through the center of the bilayer of a cubic phase and of the L, phase is indicated. The L, phase can have outer continuous bilayers outside this first shown here. F
n
Figure 12. The hypothetical membrane bilayer transition LiD F= CZD. In the L, conformation all proteins possess lateral diffusion freedom, whereas proteins located where the corresponding IPMS was sectioned (the necks with highest Gaussian curvature) become fixed in their position (F in the figure).
__
----
Figure 9. Infinite helical tubes seen along the (1 1 1) direction of the CG surface. Only the cylindrical cores of the helices are shown, and the set of rods on one side of the surface are indicated by crosses. Lysozyme
L O
2O
20
4-0
60
80
Monoolein
Figure 10. Ternary system 1ysozyme-monoolein-water2’at 40 O C . The cubic region exhibits all three phases Cp, CD,and CG.In the case of the composition 5.6%:6.6%:37.4% (w/w) of monoolein-lysozyme-water, for example, the structure is Co with unit cell axis 235 A.
phosphatidylethanolamine/high c26)should be expected to prefer the CZDconformation. A C2Dconformation associated with the spike would also “link” the action potential propagation with the mass cooperative vesicular fusion at the presynaptic membrane. In this connection it should be mentioned that the lipid composition of synaptic membranes respond to temperature changes (fish acclimated in
Figure 13. Some alternatives of a periodically curved bilayer conformation. The simplest case is shown above, with average curvature H = 0 and a symmetric bilayer. In the middle an asymmetric bilayer with H = 0 is shown, and below the most general case; an asymmetric bilayer and H = 0. Protein molecules (“free” and “fixed”) are indicated as in Figure 12.
the range 2-37 “C)and pressure changes as expected if the membrane bilayer must “balance” on the limit of an L:D + CZD transition (cf. ref 34). 11.4. Membrane Perturbations in Microorganisms: Antimicrobial Agents. There are striking similarities between the anesthetic effect discussed above and antimicrobial effects. Thus, gases which are general anesthetics, even the inert noble gases, exhibit an antimicrobial effect which correlates well with their anesthetic Moreover, it is possible to reverse this effect by hydrostatic pressure. (Adverse effects on lipid-phase transitions by temperatures and pressure discussed in section 11.1 are also seen in living cells; at 1000 bar for example cells can survive at 104 0C.62) As the antimicrobial effect of gases like N20,Xe, or C02can be related to general anesthetics, it is natural to relate the well-known antimicrobial effect of cationic surfactants to local anesthetic agents. The first type of perturbants shifts the actual transition in the direction L, cubic, whereas the second type of agent is expected to shift the transition in the opposite way. New results reported below clearly demonstrate these relations. Quaternary ammonium compounds such as surfactants have a long tradition for disinfection in the food industry and in
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(62) Enfors, S.-0.; Molin, G.Spores 1978, 7, 80.
The Journal of Physical Chemistry, Vol. 93. No. Z I , I989 7313
Feature Article
t
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80 70
~lczL-60 - .
50
0.5 1.0 CHC13-conc. in the lipid (%w/w) Figure 15. Effect of chloroform on the phase transition samples of monopalmitoylglycerol in excess of water.
L, * cubic in
Figure 14. An electron micrograph of a freeze-fractured plasma membrane of rat ciliary epithelium showing small regions (within circles) of ’orthogonal arrays of particles”, after Hirsch et aLm Magnification: X I35 000.
pharmaceuticals. Extensive studies of their effects and model of action have therefore been reported. It is clear that antimicrobial effects have been observed far below the critical micellar concentration. At higher concentration regimes, massive leakage occurs. The following two experiments using cetyltrimethylammonium bromide (CTAB) are consistent with a mechanism of activity directly related to the membrane phase transition. Phosphatidylcholine (PC) from egg yolk was dispersed in water in a concentration of 10% (w/w). Cetyltrimethylammonium bromide as a micellar solution in water was then added up to a CTAR/PC ratio of 1:2 with no detectable effects on the liquid-crystalline character of the La phase. A mixture of phosphatidic acid (PA) and phosphatidylcholine (1:lO) was also examined and found to behave in the same way as the PC dispersion. Monooleylglycerol, which exhibits a periodically curved lipid bilayer as the dominating phase,” was chosen as model for geometric effect on the cubic L, phase transition. The corresponding cubic phase can coexist with excess of water, and such a dispersion consisting of 10% (w/w) of monooleylglycerol in distilled water was used. Upon addition of CTAB as a 10%(w/w) micellar solution in water, the cubic phase was successively transformed into the lamellar (L,) phase. Already at a CTAB:monooleylglycerol ratio of 1:lO the transition was complete. This phase transition is easy to observe in the polarizing microscope due to the birefringence of the L, phase. Simple fatty acids exhibit a weak antimicrobial effect, whereas certain branched homologues exhibit quite strong effects.63 Isotetradecanoic acid, being one such example, was examined in the same model system as used in the examination of the anesthetic effect. It was then observed that this fatty acid induces an L, to cubic transition in a phosphaditylcholine liposomal dispersion. Certain polyalcohol surfactants have also been reported to exhibit considerable antimicrobial activity.& Monocaprin and monolaurin, being the most active, were examined in the same test model. No effects on the L, phase of phosphatidylcholine dispersions were observed, whereas they gave two coexisting phases-cubic + L,-when added in minor amounts to the cubic phase of monooleylglycerol. This indicates that they favor the LiD type of conformation in a membrane. The monoglyceride of lauric acid exhibits a minimum inhibitory concentration of about 5 pg/mL (studies on Vibrio paranaemo-
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(63) Luzzati, V.; Gulik, A.; DeRosa, M.; Gambacosta, A. Chem. Scr. 1%7, 278, 21 1.
(64) Reuchat, L. E. Appl. Enuiron. Microbiol. 1%0, 39, 1178.
Figure 16. Freezc-fractured lipid sample showing the change from gramicidin-induced H, to a cubic phase by t y r ~ i d i n e . ~ ’
IyticusM). We have determined the monomer concentration in distilled water in equilibrium with monolaurin crystals a t room temperature (by surface tension measurements) and found a value of 4-5 pg/mL. Thus, the concentration when self-assembly starts coincides with the concentration where biological effects are observed. From the phase properties of monolaurin itself in water it is obvious that it favors the L, conformation. There are two groups of well-known peptide antibiotics which act on the lipid bilayer. One is gramicidins, linear pentadecapeptides, and the other is tyrocidines, which are cyclic decapeptides. Both are very hydrophobic and assumed to form ionpermeable channels. Gramicidin is known to induce the L, H,phase transition in membrane lipid systems (cf. ref 66). An interesting study of the interaction between gramicidin A’ and tyrocidine (an A,B,C mixture) on a model membrane showed that they have antagonistic effects, and tyrocine-gramicidin gives a cubic str~cture.~’The texture of this phase is shown in Figure 16. It is clearly the same type of structure as shown by the CD phase. Thus, this antagonism means opposite effects on average molecular wedge shape. (Gramicidin-induced H, is “reduced” to a cubic phase.) Lunati and c o - ~ o r k e r shave ~ ~ reported most interesting phase properties of lipids from the thermoresistant organism S. solfataricus. These ether lipids form cubic phases under physiological conditions, and a membrane model with protein “plugs” similar to that of Figure 12 was pr0posed.6~ II.5. Some Observations on Plant Cell Membranes. We have recently studied membrane lipids from Brassica napus root cells, particularly with regard to effects from dehydration-acclimated plants:* A remarkable result was that the lipids formed a cubic phase with excess of water under physiological conditions. Upon
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(65) Larsson, K.; Nor& B.; Odham, G. Biochem. Pharmacol. 1912.21, 947. (66) Killian, J. A.; van den Berg, C. W.; Toumois, H.; Keur, S.; Slotboom, A. J.; van Schamnburg, G. J. M.; de Kruijff, B. Bischim. Biophys. Acra 1986, 857, 13. (67) Aranda, F. J.; de Kruijff, B. Biochim. Biophys. Acta 1988,937, 195. (68) Norberg, P.; Larsson, K.; Liljenberg, C. Submitted for publication in Protoplasma.
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The Journal of Physical Chemistry, Vol. 93, No. 21, 1989
heating, this phase transformed directly into a reversed micellar phase (an L2 phase with water aggregates in a hydrocarbon chain continuum), and this transition (slightly above 30 “C) coincides with the temperature limit of survival of the plant. X-ray data indicate that the space group is Zm3m, and the largest unit cell (maximum water swelling) showed an a axis of about 75 A. Also, after repeated water-deficit stress, a cubic phase is formed in e x w s of water, although there are differences in the phase properties compared to lipids from membranes of normally grown plants. During ripening of vegetables ethylene is produced in order to increase the respiration rate. The mechanism involved is not known, but it is believed that a nonspecific membrane lipid effect is involved, as ethylene-like effects are obtained by substances known to perturb the lipid bilayer of the membranes (for example, halogens and carbon monooxide). Ethylene has an anesthetic effect comparable to that of nitrous oxide, and it has even been used as a general anesthetic agent.69 As it induces an L, cubic lipid phase transition, it seems justified in this connection to propose that the corresponding membrane bilayer transition LzD==C2Dis the mechanism behind the stimulation of mitochondrial respiration reactions. 11.6. Some Additional Functional Aspects. The bilayer organization of the endoplasmic reticulum (ER) can be characterized by curvature and the periodicity concepts of IPMS (cf. ref 1 and 6 ) . The average curvature (H) at the rough ER appears to be zero or close to zero, whereas the H values increase along the smooth ER toward the tubular ends where the vesicles are formed. The phosphoinositide cycle involves the second messengers inositol triphosphate and diacylglycerols. Diacylglycerols result in an increase average wedge shape, and also in lipid model systems they favor cubic phases (or H,,) on behalf of L,. The inositol triphosphate initiates the release of intracellular calcium ions. Intracellular calcium ions induce phospholipid hydrolysis and immediate conversion of arachidonic acid to prostaglandins, which also will influence the bilayer curvature. It should also be mentioned that divalent ions are well-known from membrane lipid studies to induce a phase transition from La to one with wedgeshaped molecules (cf. ref 3 5 ) . Immunosuppression can be induced by high contents of w 6polyunsaturated fatty acids, which has been related to increased membrane fl~idity.’~The same type of immunosuppressive effects can be obtained by quite different lipophilic compounds, like cyclic peptides (cyclosporin A) and cationic s~rfactants.~’This indicates
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(69) Herb, I. Curr. Res. Anesth. Analg. 1923, 2, 230. (70) Wan, J. M.-F.;Tiew,C.; Babyan, V. K.; Blackbum, G. L. J . Parenter. Enteral Nutr. 1988, 12, 435. (71) Ninham, B. W.; Evans, D. F. Faraday Discuss. Chem. Soc. 1986,81, 1.
Larsson that unspecific lipid bilayer conformational effects are involved, like those discussed above. Any change in membrane fluidity should result in a shift in the L:D/CZD balance. Summary The infinite intersection-free lipid bilayer structure principle of cubic phases has been conclusively determined, and polymorphic transitions involving these phases can be explained from the average molecular geometry (cross section area at the methyl end groups in relation to that at the polar heads which defines a wedge shape). Two-dimensional sections through these cubic phases provide a possibility for a periodically curved lipid bilayer conformation in membranes. Thus, the same driving force of polymorphism as in three dimensions might operate in membranes. Literature data for the existence of such periodic conformations from ultrastructural studies of cell membranes are given. This hypothetic phase transition in membrane from the “normal” L, type of bilayer into a periodically curved one (LiD CZD)has been analyzed in cases where perturbations within the lipid bilayer favor one or the other conformation. General anesthetic agents have a strong conformational effect, favoring the CZDtype of conformation, whereas local anesthetic agents, like lidocaine, favor the LiDconformation. There are striking similarities between these effects and antimicrobial effects, from inert gases favoring C2D bilayers (including reversal effect by pressure) to cationic surfactants that favor the L, type. Available data seem to indicate that the membrane li id composition is controlled so that the bilayer is close to the LmD CZDtransition. Environmental effects on lipid compositions or effects by perturbing agents are consistent with such a control. Also, the remarkable diversity of lipid composition in membranes combined with the high specificity can be understood on the same grounds. A phase transition switch of enzymatic activity might keep this control. A methyl transferase for example could be switched on at high PE level, which favors the CZDconformation, and switched off when enough PC has been produced to give the La conformation. The phase transition mechanism has obvious advantages for cooperative membrane phenomena, such as the mass cooperative endocytosis. It should finally be mentioned that the intrinsic curvature as such can provide functional properties. Thus, the Gaussian curvature is directly related to adsorption forces.
P
Acknowledgment. I am grateful to S. Anderson and S. T. Hyde for stimulating collaboration and to I. Lindstedt for providing findings on membrane ultrastructure. Supporting grants have been obtained from the Swedish Board for Technical Development. (72) Fontell,
K. Prog. Polym. Colloid Sci., to be published.
