Cubic phases and isotropic structures formed by membrane lipids — possible biological relevance

Bi~li~mlca et Biophysica Act~ 98li (1989) 22"1 256 Elscvier

221

BBA 85348

Cubic phases and isotropic structures formed by membrane lipids possible biological relevance -

G~,ran

l indblom

and

Leif Rilfors

Deparcmenl of Physical Ch~stry. Unwe~zlyof lime& Um¢~(Sweden) (Received 3 June "1988)

Contents L

[nt~duction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

222

II,

Location of cubic liquid cl~st als in p h ~ diagraros . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

222

IlL Structur~ o f cubic ph~m:s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A, Space groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Bi~ntniuous stnmtures and p b a , ~ wilh micellar agSregate~ . . . . . . . . . . . . . . . . . . . . . . . . . . C. A~-mlnimlzlng ~ r f a ~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

225 225 228

IV. Thcorelical aspens on ~ formalion of cubic phases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A, Lipid pacldng and rpomaneous c u ~ a t u ~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Kinc6cs o| the p h a ~ tran~tions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

23~ 232 233

V.

234

E x p e ~ m e n m l methods to study cubic p h ~ and isoffopic sffuctures . . . . . . . . . . . . . . A. X-ray d i f f ~ 6 o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Time-resolved X - m y diff~lion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . .

C. Electron rmcroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. N ~ i c a r mazne6c ~ a n ~ ................................................ I. Diffusion mcoaun~ments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2, Bandsh~pe StudJ~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

E. Other s'p~t~r, copzc methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

22q

234 235

235 237 237 240

243

VI. Membrane lipid/~ater systems forming cubic phases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

243

VII. M e m b r ~ e l i p i d / ~ t ~ ss3~a~ giving jl P-NMR spectra with a n ~ o w ~ymmetrical signal . . . . . .

245

VIII, BiolOgiCal relevance of cubic phases and isotropic sw.zctures . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Membrane fusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B, Regulation o f lipid composillon in blologlcal membra~nes . . . . . . . . . . . . . . . . . . . . . . . . . . . .

248 248 249

Ahbrev~atioas: L,, norm -t mlcellar solution: L2. teve~.d mlcellar solution: L 3. bicontinuous isot~'opic p ~ a ¢ without tong-range order: L . , lamellar fiquid cryslalline phase~ HI, normal hexasonal liquid cl'ystalline phase: H u , reversed hex~$olaal liquid crysUdfin~ phase; It, cubic liquid erys~llln© phase (closed aglgcgat~); 12, cubic liquid crystalline p h ~ ( b i ~ n t i n u ~ s ) ; IPMS, infinite pexiodic m i n i ~ l surf~=; IMI, mve':ted mieetl~r intexmediate: ILA, int©damellar attschment: LIP, lipidic particic: PLB, prolamcliar body; ER. endoplasmic teticu[um: T n , ]amellar to leversed hexagonal phase transition t©mperatu~e; R0, ooontaneous radius of curvature: DSC, dlffefeatial scanning cAorimetw; EM. electron microaoopy; NMR, nt~lear ma~p~eticr~ontnce; TEM, transmission electron micto¢copy: PC, pbosphatidylcholine; DPPC, dipalmitoylphosphatidyicholine; POPC, l-palmitoyb2-dieoylphosph&tidylcholine; DOPC, dioIeoylphosphatidylcholine: DLiPC, dilinoicoy[phoshaddylcllniine; LPC, lysophosphnildylchnllne; PaLPC, l-pa]mltyol-Lpc; OILPC, I-oleoyI-Lpc; MO, t-monooleni: PE. phosphaiMylethanolamlne; Pla~ p]asmaeny}ethanolamine: GAPIaE. 81yce~l atrial o1 plasmacnylethanolamine: DDPE. dldod¢cylpho~phatldylethtnolamiae; DEPE. dielaidoylphosphaddyl. elhanolamine: DOPE, dtoleoylpho~phaddyicdianolamJne; DLiPE. dilinoicoyipht~phatidylethanolamine~ DOPE-Me. dloleoylphosphatidyl-Nm~nomethylethanolamine; DPG, diph¢6phalidylg]ycefoh P~. pbc~pliandyl$1~¢erol: PS. phosphatldylserine; PI, phosphs~xdylin~itol; MGIuDG, raonoblucolsyldiacylglycerol; M G ~ I D G ~..oat.ip0a~tosyldiacylSlyt~rol , DGIuDG, dislucosyldiacyl$1yccxol; DGalDG, digvJacl~yldiacylglycem]; DOMGIaDG~ rl~.!aoy [n..onogluc~syldia~ylSlyceml; DODGIuDG. diole0yldi~uc~syldiacylglycerol; G D N T , glyccroldia[kylm,nitol tetracth~; GL, a rmxtm'e of: "/0~o or G D N T with fl-D-Slycopyran~ linked to the nonitoi group: 30% of g]~erot d~alkyl gl~,~.erol tetraether with ~.~.sa~ac. t opy~an~yl-~-D-sal ~ topyranose linked to one of the glycerol groups. C o n r . x p o n d e ~ : G. l.J,ndblom. Deparm~nl of Physicni Chemistry. ~Jnivcr,Aty of Urn©h. S-901 87 U m c ~ Sweden. 03044157/'89/$03,50 © "989 ~lsevi~-r Science I~.lblishers B.V. (Biomedical Division)

222 Membrane=lructurcs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D F~*I dig~sti~ . . . . . . . E. Activilyof m©mbrane.bound¢~ymcs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

251 252 253

C~,~u~i~',

253

C.

IX.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Ae~-,,wL-"~ge~nls Ref©ren~

I.

....................................................

...................................................................

Introduction

Membrane lipids like other amphiphilic molecules can form a variety of different lyotropic liquid crystallille phases. Among these, the lamellar phase with its muhibilayer structure has long been well known to the biologist. However. the awareness of the formation of nonlamellar stpacl~tres, occurring for a number of mum. brunn lipids, has gradually changed the view of many biologists on the functional role played by membrane lipids in cell processes. It is no longer believed that lipids form an inert matrix, and only thai. where the important prot,~lns are incorporated. ( I n the c o n t r a r y , there is n o w a great deal of experimental evidence showing that the lipids actively par',icipate in many important functions of the cell. and part of the aim of this review is to call the attention to such events and in particulm to the role played by t h e class of liquid crysz~:l~r,= structur¢~ that have a cubic symmetry. These lyotropic liquid crystals exhibit, like the lamellar and hexagonal phases, long.range order but have disordered acyi chains. The a~.regates formed by the lipid molecules are situated in a cubic three-dimensional lattice. Scwcral different cubic structures exist, as will be dis. cussed in this review. I n contrast to the lamellar and hexagonal phases, which are optically birefringent, the cuhiu phases are optically isolrc*pic. Due to this isotropy cubic phases may in some cases be difficult to discover experimentally. Furthermore. they arc very viscous. often waxy or in some cases jelly-like. They can also be extremely melastable; for example, one of the authors had a phosphatidylcholine sample forming a cubic phase ~t about 6 0 ° C in his drawer (usually at room temperature) for several months :znd the sample still stayed cubic. The first observations of cubic phases in membrane lipid/water systems were made in the 1960s. However. it is not until recently that m o r e intense altcution has been payed to these phases and that a greater knowledge of cubic phases has been accumulated. For simple surfactams and soaps several reviews arc available [ I 4] where cubic phases are discussed. In ord~r to keep this review to a reasonable size we restrict otzr discussion to systems cot:~fi:ting membrane lipids, b.:ing convinced that readers ol this jolarnal will appreciate sucE a restriction. The review includes a critical discussion of the

253 254

physical methods used for studies of cubic phase SlrUC. turcs. This is especially important when studying these phases, since several methods are required for an unambiguous determination of the phase structures. There is certainly a need for methodological developments. Nuclear magnetic resonance INMR) has proved to be a very useful technique in many respects, Furthermore. transmission electron microscopy (TEMI, recently applied to membrane lipid systems, is a promising candidate. A clas.~ification of the different cubic space groups used in X-ray diffracrion is also briefly discussed and their most common acronyms are given. This has been done since it is not always clear to a reader of the literature in this field what they really mean. The theoretical treatment of the formation and structure of cubic phases has advanced considerably over the last few years, It has been possible mainly due to the applk-ation of differential geometry to the de,'scription of the complicated surface~ building up many cubic sreuctures. This approach is also presented in this review. T o Ibe best of our knowledge this is the first at~,.-'m.~', made k; try to collect all data published ~o far about tncmbrane lipids involved in the formation of cubic phases. I I. Lecttlen ef ¢ubk liquid eryssels ia pltase 4 ~ Amphiphiles or lipids may be divided into two groups: (I) water-soluble micelle-fotming amphiphiles: and (2) water-insoluble swelling amphiphiles. An example of the first group is lysophosphafidykholin¢ while phosphatidylcholine belongs to the second group. However, there is not always a sharp boundary between the two groups, since an insoluble swellin8 lipid at room temperature may be soluble and form micelles at a higher temperature. Cubic phases form in several membrane lipid systems and their existence often critically depend on temperalure and composition. A first step in the investigation of membrane lipids must therefore be to derermin¢ the phase diagram of the lipid/water system. A limited number of comF'~:te or partial diagrams of that kind are available in the literawr¢, some of which are discussed in this review. It should also he ,'aemioncd that reviews containing detailed phase diagrams for a large number of simple surfactants have been written by several authors (see e.g. Refs. 2, 5 and 6). D¢tcrmination~ of

A

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Fi~S~n~c~ure~.di~rcr~n~ani~.c~c~lq~d~r~s~a~ln~.ph~ha~mem~rane~d`~an~`~;~ ,'s N¢~n..a hcxa~,,.~ i pha~ ~B at~ at (L.) phase; and ((') ~-vCrst.d he~,t~n~; (lIlt) pha~¢ N,~I¢ Ih¢ ~an~u, ,l~pt~ ,d the lipid el~,]~'~ll~ In~rft~mj to Ih¢ flguf~-nI-k~.4~l h~urc ~daplcd rr,~m ReI 1(~ phase diagrams a r c u s u a l l y vcr~ ledic, un w o r k u.~ing classical m©lhods. T h c n c ~ l [ o r m o r ~ ~a~" ~ ~ c ~ h ~ l s I ~ |o t l ~ d e v e l o p m e n t o f N M R techniques:~,ch i n v e s l i g a l i o n s [?.8]. T l ~ ¢|¢ganl a n d c e n s ~ z . : n t m c l h -

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qucnlly mil~cd in our |a~ralory [9 121. The mos~ ~bundanl pha~cs [ound l'or the 3mnhiphi[ic syslcm~ o1" ,zlcrcst hcrc ~lrc: {I) the miccllar ~ l u l l o n wish n(~rmal

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224

IL l) or reversed (L:) aggregate structure:,; (2) lamellar IL~) liquid crystalline phases; (3) normal (H.) or re¢ersed (HI0 hexagonal liquid crystalline phases; and (4) numhe~ - of different cuhie liquid crystalline phases. the structures of tl- ~ aggregates in groups 1-3 are well :stab[fished (cf. Fig. 1) and wc will no" deal with them further, but the structure of many of th~ cubic phases is ;till under debate and will be discussed in later sections. l h e positions in the ; h ~ e diagram of the various ~bases occurring in a lipid/wate~ system often give cery helpful info.~afion about the physicochemical ~ropcrties and, in particular for cubic phases, about the lggregate struc, ure building up the phases. Hitherto, a :ubic phase has been observed between almost every 3ther phase in the phase diagram. This will be exemplified in the following, starting at the high water conrent side of the phase diagram of a soluble micene-for~ing lipid. In a binary system containing lysophosphaLidylcholine (LPC) having saturated acyl chains with 12, 14 or 16 carbons, a cubic (It) phase exists between the ~cellar solution and the H I phase (Fig. 2) [10,13]. If a • ,ird component like an alkane or a soap or fatty acad is ldded to the cubic I 1 phase of 1-palmitoyl-LPC :Pal,PC)/water it disappears from the ternary phase :liagram at very low additive contents (Ref. 14 and Lindblom, O, and Pdlfors, L , unpublished resuhs). Usually, monoaeyl chain lipids like LPCs form micellar ~ohitions but there is an interesting exception, namely ~he swelling amphiphiles of monoacylglyceroles, several ~f which form ~ubie phases in equilibrium with an aqueous solution (Figs. 3 and 4) [15,16]. For micellar. forming systems, cubic phase areas occur most freq~emiy ~et~een the Hx and the L~ phases, as for ~xample in u n s a s m ~ I , p c systems (Fi 8. 2). Cubic phases for binary systems containing a swellmg amphiphile usually form in equilibrium with an L , phase on one side and with an excess of water on the

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~ t ./.(,/,I,ahel Fig 4 Pb~c diagram for the system MO/H;~O The biconfinuo~ (f2) ~uhlc phases are denoted G and El (se: sabs~fi~:a II1CI L=. r~crscd miccllarsolulionphase Ol~cr no~tions accordinggolegends or FigS. 2 told 3. FrOm Per. 28.

other side (for example monoolein; Fig. 4). Another possibility is that the cubic phase is in eqJilibrium with an H . phase, which in turn ~ in eqinlibfium with excess water. The latter case is found for mouoglucosyldiacylglyeerol (MGIuDG) from Acholepg:;.snm la~dlawii membranes [11] (Fig. 5). For ~'¢¢-anmponant systems it has been found that cubic p~ases form at different locations in the phase diagram in the same way as described above. Ca0ie phases of membrane lipids often for~i between L~ and H u phases. Some ternaiy phase diagrams are shown in Figs. 3, 6 and 7: 1.monoolein ( M O)/dioleoy Iphosphatidylcholine (DOPC)/water. egg PC/sodium cholate/wat~; and "; monogalactosyldiaeylglycerol (MOallX~)/digalactosyldiacylglycerol (DGalDG)/waser. It has also been observed that for s o . ~ ~y~icms isotropic phases, probably

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Fig. 3. T¢n~five I©rnary phase diagram for the sysIcm M O / D O P C /

2H20 aL 2S°C. L2, i~tropi¢ phase; HIi , r~r~e~ hexagonal Uquid crystalline phase. Other notations according to legend of Fig. 2. The diagramis adapted from gel re.

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225 Sodium

cholate

while the lamellar and hexagonal p h a s ~ are anisotrop~c. However, the cubit phases, like other liquid crystalline phases, hate no short-range order, i.e., the hydrocarbon chains are disordered.

Ill-d. Space groups

W~ter L~°ithi~ Fi8. ~;, P h ~ d i a g r ~ for the system ~ Tolk PC/sodlum cholate/HzO at 22aC. Notations according to legend ot Fi~ 2. Redrawn from Ref. 180.

of cubtc structure, from as metastable interntediares at the thermal Fhase transistion between the L . and H u phases [17,18]. Finally, it is important to note that so far a cubic phase has never been observed between the H n and the L 2 phases. 111. Stmettm~ o# enhi¢ phases A cubic llqmd crystalline lipid/water phase is one in which the lipid aggregates form a three-dimensional lattice, The lipid aggregate units can have different shapes such as spheres, rods or lamellan (Figs. 8 and 9). In contrast to cubic phases, lamellar liquid crystalline phases exhibit a one-dimenslonal peri~-~Jicity, i.e. lam..eIlat units of infinite extension are stacked regularly (l:;,, IB), and hnvr~onal liquid crystalline phases exhibit a two-dimensional periodicity with rod-like aggregates of infinite length packed into a hexagonal lattice (Fig. IA and 1C). Thus, the cubic phases are optically isotropic MGD6

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2820 at -10°C, Nota:i~,J~accotdth8 In legends of Figs, 2 and 3, From Ref. ¢, and Selslam, E~ nreaml, I. and IAndbl~, O., unpublished data.

The cubic phases are classified ~ccording to their space group obtained by X-ray diffr~£6on. The number of different cubic s'..'~,:tur~ might hr quite Izgn, but the space groups carefully dm~nnined for cubic phases c:,,i. raining membrane lipids are still limited to a ~ew. We will only discuss the latter here and for other amphiphile systems the reader is ,'efcrred to hhe :-.vlews [}-4.1. Thus the characteristics of the following space groups will be considered and they are denoted according to wbetber the cubic: structure is primitive (P), body-centred (1) ('inncnzcnnum" in G~man), or face-centred ,~F) (Fig. 10). A lattice formed by those aggt~egates which can be superimposed thrcugh p~d~el translatinng (i.e., without rotation or mirror re/iecuon) is a B:aoais lattice [19], There aye fourteen different kinds oi" "3~avais iaitices, but here we only consider those for the cubic lattices P, F and L Generally, for a crystal structure, transformations in which a w~-~ecular configuration ts superimposed onto the origma! configuration are called qymmetry t~ansfr.rmafions. When a m i t e r reflection with respect b') a plane is involved in the symmetry transforraatioa, ibis plane is calhxl a mirror plane, and will therefore be denoted by the symbol m. When a rotation through an angle 2~/n around an axis is included in the symmetry transformation, that axis will be called an axis o/rotefinal symmetry of the n th order, or an n-fold axis. Tb.i* is denoted by "2. 3 . . . corresponding to n = 2, 3 . . . When a combination of a rot~th:n th:,mgh an angle 2win around an axis and an inversion with respect to a point on the ,~.xis is included in the symmetry transbrma!ions, that axis is called a rotater2"-mvErsion axis of the nth order and will be denoted by 1, 3, 4. etc. (2 is equivalent to a mirror plane) corresponding to n = 1, 3, 4, etc. When a combination of a translation through a certain distance parallel to a plan..~, and a mirror reflection with r¢~p~'ct to the phinc is ~uchided in the symmetry transformation, that plane is called a glide.refl~don plane. When the parallel translation i~ in t,he direction e.r the a-axis and the translational distance is equal to haft of the period, it is then called the a-glide-reflection pit nn and is denoted by the letter a, and the same applies In the b- and c-axis. When the l,,arallcl translation is in the direction of the diagonal lizte of a face and its I ranslational distance is equal to 1 / 4 of the diagonal period, it is called a diagonal glide-reflection plane or the diamond glide-reflectlon plane. It appears in the crystal structure of diamond and is denoted by the

226

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~

228 symbol d. The space group completely represents the symmetries of the liquid crystalline phase structure, la3d This body-centred space group gives an X-ray diffraetogram with spacing ratios equal to ¢'3-: Vc4: ~/ff: ~ : ~ i o : f'iT etc. Cubic phases with this symmetry seem to be one of the most commonly observed in lipid systems. It was the first cubic space group for an amphiphile system that was unambiguously determined [20]. This particular system contained an anhydrous strontium soap at about 235°C, where 17 reflections were observed in the X-ray diffractogram. Since then, similar X-ray results but with much less reflections have be~J o b t a ; n ~ for a number of liquid crystalline ~5':" terns, having phase areas in between Lo and H n phases and in between L , and H I phases. Luzzati and Spegt [20] proposed a structure for the cubic phase in whit,h rod-like aggregates are joined three by three to form two interwoven but otherwise independent three-dimensional networks. In most membrane lipid systems, htswever, a different description was recently shown to be more appropriate, namely the use of so-called areaminimizing sl~rfaees, as will be described in subsection III-C. Pn3m. This is a primitive cubic lattice. The spacing ratios are ~f2~: ~/3: v~: 1/6: ~/8: ~fr: 1~O: etc. A cubic structure with this space group was first proi~ased by Tardice [21], who suggested a struet:) z built up by reds connected four by four at tetrahedrll angles, buildihg two unconnected but interuoven diamon.i latticee. More recently it has been suggestf: l that cubic phases belonging to this space group, at least those with membrane lipids, are better described by minimal surfaces. Prn3n. A primitive cubic lattice having the spacing ratios ~/2: v'4: ~/5: ~/o : ~fff: etc. Cubic phases with this structure have been found to be loe,~ed between tile aqueous L I micellar solution and the H I phase. [t has been obser,,ed in many binary and terp.ary systems. Tardien and Luzzati [22] proposed a sir,notate for these cubic phases to be built up of a ~hree-dimensional network of rods to form a cage in which a spherical mice!!e is enclosed. However, recently this structure propo'::,i was shown to be obviousl:/incerrect by Eriksson et at. [13.23] - it is in fact in total disagreement with N M R data - and instead a structure composed of short rod-like micelles (Fig. 8) has been suggested for this phase (subsection V-DI. It should also be mentioned flint, in particular re, this cubic phase occurring at high water conte~l~, the number of small-angle reflections are often so few that it is almost impossible to de'.ermine the structure unambiguously. In many cases only two spacing ratios of s ~ : ~/4 are observed and even a face-centred space group has been suggested for cubic phases with this location in the phase diagram (see the renew by Fontell [3]). s ~ , . t~ body centred cubic lattice with the spacing ratios ~/2: vt4:~/6 : ¢8: ¢'lv: q ~ : v~'~ etc. ~/o tar this

structure has not been unambiguously shown to occur but it has been suggested to be a possible candidate for several lipid systems [18,24,25]. In a previous paper by Lindblom el el. [26] it was suggested that a cubic phase of the system M e / w a t e r had an Im3m structure. However, later Longley and Mclntosh [27] showed that an isotropic F.quid crystalline sample of this system at high water contents belongs to the space group Pn3m mr Pn3. This initiated Hyde ct aL [28] to investigate in more detail the phase equilibria -ff the M e system, where they found that the previous cubic phase area in the diagram corresponds in fact to two cubic phase structures, namely la3d at low water contents and P n 3 m / P n 3 at high water contents (Fig. 4). However, as will be discussed below, the N M R diffusion investigations, which are a main part of the paper by Lindbhim et al. [26], are not affected by )he problems with the interpretation of the X-ray data. Further investigations of systems asserted to have the l m 3 m structure are currently in progress. Other .~pace groups. Recently it was shown in a careful X-ray study [29] that a lipid system containing an enzyr~e, MO/cytochrume c/water, forms a cubic phes¢ having PZ I - - symmetry (denoted Q212 in Ref. 29). This space group is non-centrosymmetric and it was pointed out that this is the first unesluiencal example of a lipid-containing phase with disordered acyl chain~ and a ehiral structure. The cause of the chire!ity must be sought in the protein molecules. In the structure of this cubic phase it is proposed that part of the lipid network is replaced by a set of identical globules; the u.~t ccli contains four such globules. Most probably sever~il other cubic structures exist, as for example Fd3m, but there are no sufficient ¢xperimental evidence for tlmse at prescllt. III.B. Bicontinuous struer4r~,x and phases with micellar aggregatea From a methodological point of view it is convenient to divide the cubic phases into two fundamentally different groups: (1) cubic structures having regions which are continuous with respect to both polar (water) and nonpolar (hydrocarbon) compuncnts; and (2) cubic structures built up either with discontinuous hydrocarbon regions but with continuous water segions (for example normal micellar aggregates in water) or with discontinuous water regions but with continuous hydrocarbon regions (for example reversed micellar aggregates in hydrocarbon), Cubic phases belonging Io the first group are usually called bicont#~uous, since they are coutinanus in both water and hydrocarbon. Most of the cubic phases formed by membrane lipid~ have uoc,,a shown to have such a str~lc)ur~ [30]. ~So far a c,a~ic Fha~e buill up of reversed micelles has not been e~perimentally found.

229

-

We have observed that there is a ren~arkable difference in viscosity between the two types J[ struclures all the bicontinuous phases are extremely viscous while the phases built up of globular aggregates usually are less x'iscous ann have a more icily-like consistence. Furthermore. the hlcontinuous phases exhibit a more hysteretic behaviour.

111-C. Area-rainimizing surfaces It was first suggested by Striven [31] that bicontinuous cubic phase structures can be described by periodic minimal surfaces, well known in differential geometry [32,33]. In order to fully appreciate the use of the mathematical principles of differential geometry in the description of cubic phase structures the reader is recommended to consult one of the many textbooks on the subject [34,35]. A thorough discussion of such a complieated theory is outside the scope of this review. Here we will only give a brief account of the most impo.rtant concepts and definitions, sufficient to get a rough understanding of the results obtained, In particular it is important to grasp the meaning of the class of mathematical surfaces known as periodic minimal surfaces, often referred to as IPMS (infinite periodic minimal surface). A minintal surface is defined to he a surface having at each point a mean curvatm'e of zero. What is mnam by curvature? Consider the normal N emanating from position P on a surface. N will point from P to the cet~.er of the best-fitting circle tangentially touching the surface at P, or it will point in the opposite direction. If it points into the circle, the curvature K is equal to l / R . where R is the radius; if it point~ away i ¢ = - l / R . Thus. ~ ~.~m be positive or negative, dependip~ on the choice of tile normal N. A negative ~c meaos that a curve through P curves away ,rom N, and a positive g means that it curves towards N. Two measures of curvature are used in differential geometry: the Gaussian curvature K ~ ~:l~:? and the taean curvature H ~ I/2(K t + ~2), where Kt and ~2 are the two principal curvatures. That is. the surfaces of a minimal area satisfy H = 0, so that every point on a minimal surface is a balanced saddle point with ~l = - ~ 2 . Consider now a simple example of a physical interpretation of minimal sl~rfaces. We can relate the pressure differenet: Zip between the sides of a soap film at any point to the mean curvature of the film by the Young-Laplace equation ~36]: Ap =7t1. where ¥ is the surface tension of the soap liquid. The same equation relates the mean curvature of art interfacing surface between two liquids ~o the difference in tLe pressure betwt~n them. Thus, b," = 0 means that the pressure is the same on both ¢ides o. a alie.lma! surface. A soap film meeting these requiremc ~tq can be obtained experi._m_e~t-Ily by dlt~=dnoo two e l o - d wtre~ or coaxial rings into a soap solution and then withdraw them. If we are

lucky, we migh,t see one connected soap film hounded by both wires having the shape of a so-called catenoid (discovered by Euler in 1744). The connected minimal surface wdl not form if the two circles are too far apart. but only a certain distance between the rings will give a stable connected soap film. For this film it is easy to see that the pressure is the same on both sides of the film alld that two curvatures exist differing in sign. i.e.. at each point the surface is curving both away and towards a given ,-_erpendicular direction and the minimal surface looks like a saddle surface. In the interior of a soap bubble, on the other hand. the pressure is higher th",m on the outside of the bubble; thus a bubble is a surface of constant-mean-curvature H :# 0. Schwarz discovered in 1865 principles that allow t:s to build up infinitely extended minimal surfaces without self-intersections from sma!i pieces. Such an object is called a ~erwdic minintal surface. For a liquid erystelline phase the simplest example of such a surface is the plane, as in the periodical organization of a lamellar phase, where bilayers of amphiphiles and water are alternatively stacked with fiat interfaces (Fig. 1B). Thus. the lamellar phase can be seen mathematically as a volume divided into two subvolumes without self-intersections, each of them physically continuous (water and lipid bilayers). In this case we have a periedic~ty along one dimension, but an IPMS may also he continuously extended throughout the space. Such surfaces of cubic symmetry, and their constant-mean-curvature relatives. have been successfully used to describe the structure of reversed bicontinuous cubic phases in both lyotropic liquid crystalline systems [28] and block polymer systems [37]. The MO/water system exhibits two cubic phases in equilibrium with each other [281 (Fig. 4); one with a body-centred lattice at low water contents and one with a primitive lattice [27] at high water contents. Both these structures have been pro?osed to be described by IPMSs. The s.t-~ee group Pn3m for the primitive lattice corresponds to one of the fundamental cubic IPMSS. namely the "diamond" type (D), and the group la3d corresponds to the "gytoid' type (G). A third well known IPMS is the so.called Schwarz' surface (P, 'primiti~z') corre~i~o!~cv~g;~, ~ . space group lm3m (Fig. 9). The structure of this hicontinuous reversed enbic phase is formed hy an infinite lipid bilayer, ,J'hich is arranged so that the Schwarz' surface is draped on either side with lipid monolayers whose terminal methyl groups of the hydrocarbon chains touch on the surface. A description of the cubic phase structure by IPMSs is, no doubt, very fruitful as will be seen in the eollowing. The transition between the two cubic phases ~urmed in the MO/water system can be understood by oar.,siderlng a transformation (a so called Bonnet ~ransformation) fzom a G- to a D-surface [28]. This means that a phase tra,tsition occurs between ,wo lipid bilnyer structures, witr,out any change in tae average .eurtature,

230

..~ 10 ~E

euble

,

L

30

31

I[

32

33

34

10=/T (K1)

103/T (K')I

Fig. 11. Lipidtranslationaldiffusion ~fficients in lame.liarand cubic phases. (a) MO/I HzO (12 ~t% zH20). The stmctu~ of the cubic pba~ is ~sumed to be built up of bilayer units. (b) Samplewith a DOPC/DOPEmolar ratio of 1.0 and with tO wt% :ZH20.Filled symbols refer to the rn~ured diffusion coefflclents and open symbols to the calculated I~al dlff~ion e¢~-_fli¢ienls in a bicontinuous cubic phase consislingof bilayer units ((>) and rod-shapea unils(o). From Rcfs. 26 and 30. suggesting v e ~ small changes in the enthalpy of the transition. Hyde et al. [28] also found by differential scanning calorimetry (DSC) studies that tilt cnthalpy of the transition was less then about 0.01 IO. mol-l. This value shou!d be compared with the phase trans,~tion enthalpy of about 1 kJ. tool -1 found for the transition between the cubic and the H u phases formed by this system [281. A bi!ayer structure in these cubic phases is also strongly supported by our N M R diffusion studies of lipid anlf water. Thus, it was found (subsection V-D) that the lipid lateral diffu~io~ coefficient directly measured in the lamellar phase compares exactly with a local diffusion coefficient for a cubic phase composed ..~ h!layer units in the MO/water system [26]. Analogous results [301 are obtained for a phospholipid system compos=~d of DOPE/DOPC/water, where it also was possible to orient the lamellar phase at room temperat~re and study the translational diffmlon as the system

transformed to a bicontinuous cubic phase at increasing temperatures (Fig. 11). Recent investigations of w~ter diffusion coefficients as a function of the water content in the reversed cubic phases of the M O / w a t e r system (Erikssono P.-O. and Lindblora, G., unpublished data) strongly support a structure described by a minimal surface. As can be seen there is a discontinuity in the water diffusion coefficient (Fig. 12b), hut not in the lipid diffusion coefficient (Fig. 12a), at the water concentration where the transition between the la3d and Pn3m cubic phases occurs. Anderson (personal ¢ommuincation) found by solving the diffusion equation that a change from a body-centred symmetry, to a primitive one (calculations were done for four different symmetries), while keeping the volume fractions the same, the values of DH=o/D~o, computed by using IPMSs, change significantly with the change in symmetry. Here DH=o is the wat¢.t diffusion coefficient of the cubic phase and D~2 o

04 I b

~)6 a ~,.

I

o

I

/

lo

o. o

s

10

15

5

to

lg

• H2o/np~d (moz/mol} H ; O / lipid ( m o l / m o ) l FiG, 12. The lipid diffusion coefficicm Ca)and the water diffusion coefficient (b) in the cubic ph~e or MO/water as a function of the water conical

at 25° C. DH2o rcpr~ents the diffusion coefficientof water in the cubic liquid crystallinephu~e and D~lao is the dlr'lslon coefficient of pure water. From Eriksson. P.-O. and Lindblom. G.. unpublished data.

231 is the water diffusion coefficient of bulk water. However, by using the interconnected rod-model of Luzzati and co-workers for the reversed phase structure, DH2o/D~7odo not change. It is interesting to note here that the water d:ffusion coefficients obtained For three other lipid systems at high water contents, forming cubic phases with a primitive space group (Table It in get. 30), have mmilar values of the ratio DH,o/D~= o as those ~btained in the corresponding MO system. Furthemlore, it can be inferred from Table i that at low water cont,o.nts, "always cubic pli~es belonging to thu la3d space group are formed, while at high water contents cubic phases with a Bn3m symmetry are observed (and in some cases at even higher v,-a:er contents cubic phases having lm3m symmetry are formed). Co~,sidering the fact that the ratio of the lattice parameters during the minimal surface transformation (Bonnet), which preserves surface area, are 1.00:1.022:1.07 for the G, D and P surfaces [28], it is not surprising that the reversed cubic phases observed are formed in that order with increasing water content. Cubic phases having G, D and P surfaces are formed in the same order, also when a constant-mean-curvature calculation is performed. Although IPMSs have proven veG, ~qefni in descrth. • ing the symmetry and topology of bicontinuous cu~ie phase microstsacturas, this still leaves open the question of the exact shape of the surfact~ describing the p o l a r / apolar interface. In bicantinuous cubic phases of the normal type, such as the la3d cubic phase occurring between the H~ and the L= phases, a minimal surface such as the "gyroid" is the imaginary midsarface placed in the aqueous continuum, while the two lipid c~etworks are each threaded by a graph (called a 'skeletal graph" by Schoen [38D. Each edfe of such a graph defines the centre-line of a lipid region, and since the length of the lipid is ;elatively constant, the lipid/water interface should be well described by the interconnected rod-network model that Luzzati and colleagues have proposcti, whioh satisfies both constant-length ~ d ,:ontant-meancurvature at the interface. N M R diffusiotl d a m on this lype of cubic phases are also consistent with this model, where the lateral diffusion coefficient and the diffusion coefficient in the cubic phase usually differ by a factor of three or more; for example for OILPC the ratio between D L and D,~b is about 3.8 [13] (subsection V-D). However, for the more common ret~eesed biconlinuous cubic phases, the appropriate IPMS descrthes the midsarface of the lipid bilayers (Plate I b g and in such a case the c lstant-]ength (see below) and constantmean-curva'cre (Plate la) (for Plate I see page 226) interracial s~ ;faces do not cnincid~ Reccmtly, five families of constaat-mean-curvat ure surfaces exhibiting cubic symme:ry have been tracked, and applied to the interpretation of data from cubic phases in surfaetant [39,40]

Theory

Experimen

Fig. 13. A split-semen photogr~ with TEM data from a bl~k co~olyraer ¢ubk phase on the lower half, add a conjurer simuhdon using a cOnSlanbmean-cm~ai~ alodcl on the top half. The simttlation was o0mpmed by scndina rays th,~gh the Iheov:liealmodel and calculating the resulting plxel densily on ¢be image plane. The mulch between [be microscopy data and the s/melation provides convin¢mg~sual evidenceof the constant-mean-curvatu.~ imedacial surface. By¢ourlesyof Dr. IX Andes--. and block copolymee systems ]37]o Surfaces parallel (constant-length) to minimal surfaces (Plate I,:) have also been investigated as possible interracial sur f;ices. In a comparison of the two types of model surface (l~arallel and constanbmean-curvamre) for the case o f Pn3m symmetry, it has been shown [411 that while th~ variance of the mean curvatures over the parallel sur[ace is typically 20-30~ of the square of the averagb~ the variance of the lipid length using the constant-meancurvature interracial surface is much smaller, typically less than 1% of the squared average (se~ also subsection IV-A). In all probabigty the surface minimizing the sum of the curvature and stretclihtg energies lies somewhere between the constant-meon-cervamre and the parallel surface. Recently, bicontinnous phases of cubic symmetry have been found in block c,:polymers [42l, where TEM series experiments provide a means to verify the microstructure directly. Model stractar~es defined by surfaces of constant-mean-curvature with PnJm symmetry have been used to produce remarkable matches between TEM micrographs and computer simulations (Fig- 13). Mathematically, surfaces of constant-mean-corvature minimize the surface area under the constraint of given volume fractions, and in a thermodynamic treatment of so-called star dlblock morphology [37], the constantmean-curvature structure has been shown to lie favoured over either interconnected-rod or parallel surface structures. IV, Theoretical aspects on the formation of cubic #roses

A complete theory of the phase equilibria of m e m brane lipid systems must he able to give an explanation

232 at the molecular level of the formation of the different liquid crystalline phases, I, Lo or H u, as a function of concentrationl temperature, pressure etc. The development of such a theory has to begin with an understanding of the factors leading to the formation of aggregates of different shapes (spheres, rods and lamellae) building up the ordered phase structures, and how the separation of different phases occurs upon interactions between the aggregates a n d / o r aggregate growth. Although there is no theoretical model coveting all the different situations occurring in lipid/water systems, theoretical treatments of a certain kind or class of lipids or lipid phase structures have been very successful. In particular for ionic and zwitterionic lipids, where the dectrostatic interactions play a dominant role, WennerstrOm and co-workers [43] construcled a phase diagram of for example dipalmitoylphosphalidylcholine (DPPC) and water, which is in excellent agreement with the experimentally determined one 17]. However, no cubic phase is present in this diagram.

IV-A. Lipid packing and spontaneous curvature One of the most useful concepts for a qualitative understanding of the phase bchaviour in amphiphile systems is based on a consideration of the shape of the lipid molecules (of. Fig. 1), first suggested almost 35 years ago by Tartar [44] and later developed by Tanford [45] and by lsraelachvili and co-workers [46]. The selfassembly of lipid molecules depends on a dimensionless packing parameter defined by the ratio o(al) -1, where v is the molecular volume, 1 the molecular length and a is the polar head group cross-sectional area. When the packing parat~-eter is equal to unity (cylindrical-like molecules, cf. Fig 1) thexe are optimal conditions for the formation of a hilayer z:~cture, while for v[ai) "t > 1 [46.47] the m~flecules are wedge-shaped and the lipid monolayer p;efers to curve towards the water region, i.e., an H n phase may form (Fig. 1). Although this simple approac/t is very useful for qualitative considerations, i t is difficult to use it for more quantitative calculations, mainly due to the complex dependence of a on temperature and concentration. Furthermore. a change in the molecular shape does not explain why an H . phase is formed at high water contents in PC-systems when an alkane or hydrophobic peptide is added [12,17,48]. This problem was nicely solved by Gruner [4o], who used. a concept related ~o the packing parameter v(al) -1, that has a more general character na mdy the so-called spontaneous curvatu,e of the lipid monolayer H o (cf. subsection III-C). This was first introduced by Hclfrich [50] for PC bilaycr systems, In the work of Gruner and co-workers [49] it is shown that the energy of curvature plays a dominant role in the formation of H n phases. The total curvature free energy,

Gco~c, per area, -4, is given by [41]: ~o

= Kma
where H is the local mean curvature of the monokiyers. and K m is the elastic bending constant. It can thus be seen that it is advantageous to have H close to H o to minimize the free energy of curvature. However, for a phospholipid monolayer to form a cylinder of radius Ro ffi 1/tlo there will also be a cost of nonzero packing energy [49]. In particular, the acyl chains of the PC molecules must stretch to cover the regions in the middle of the proximate water cylinders in the Hit phase.. We thus have a situation where two physical forces, curvature and packing, appose, and such a situation is leferred to as a 'frustration' [51]. It has been shown in several studies [t2,17,49,52] that this 'frustration' can be decreased or eliminated by the addition of hydrophobic molecules. In ~n H u phase, N M R studies [12] strongly indicate that these molecules preferentially partition into the hydrocarbon regions in the middle between the water cylinders. The obvious question now arise: can the same tbeoretical approach also be used for an understanding of the formation of the bicontinuous cubic phases? Attempts in this direction have been taken by Charvolin [51,~3] and recently by Anderson et aL [41]. As discuss~:d in subsection III-C, the bicontlnuons tunic phase structure, in which monolayers are draped on each side over a minimal surface, this surface describes the midplane of the bilayers and not the interface between the polar and the hydrophobic regions (Plate Ib). Thus, it describes the location of the terminal methyl groups of the lipid acyl cha(n~. For the cubic phases the actual dividing surface is displaced from the minimal surface by the length of the acyl chain on both sides of the minimal surface (Plate le). Therefoce cubic phases built up of hilayers should not be referred to as having a zero mean curvature interface. It has been shown that the requirement of symmetry with ~espect to the two halves of die brayer, leads directly to minimal surfaces as midplane surfaces, and through a construction involving projections of surfaces in four-dlmensional space leads to the minimal surface, which describes the family of bilayer cubic phase structures [51] observed in experiments. Recently, differential geometry wa~ applied Lc, bicontinuous cubic phases composed of bilayer units, and dfis problem has been treated quantitatively [41]. The idea was to delermine whether a cubic phase can be a solution to the problem of frustration (of. H n phases), i.e., whether the cubic phase structure can have a conslant-mean-curvature (at its preferred value of H0) and at the same time keep a coflstanl thickness of the bilayers. It was shown that these two energy terms are frustrated and cannot be satisfied shnuhaneously. How-

233

ever, it was found that the "frustration' can he smaller for the cubic phase than for the L~ and H n structures. It should be noted that the cubic phase u~oally appears between the L~ and H u phases in the phase diagrams. It has consequently been shown that curvature plays an important role ,*or ~he stability of reversed bicontinuous cubic phases, and that also other interactions are present which have to be included in a satisL~:o.~, description of the phase, The interactions between the interfaces or the aggregate units building up the cubic structure must be introduced into the calculation (hydration forces, hydrogen bonds, Van der Wa,als forces, ete) as well as a good description of the chain packing entropy [41]. Another strong argument for the fact that curvature is important for the formation of tbe~e cubic phase structures was recently reported (Anderson. D., Wennerstrbm, H. and OIsson, U., unpublished data), who quantitatively calculated the position in the phase diagram of the so-called L~ phases, having a structure closely related to the cubic phases. The isotropie L~ phase is observe~ especially in many nomonic amphlphile/warer systems and it is generally in eqaifibrlum with both a dilute solution and an L~ phase and has a very narrow stability range. The latter often pertains also to the cubic phases The basic structural unit ha the L 3 phase, like in many cubic phases, is a lipid bilayer. Anderson ¢t at. showed that the drivin~ force for the formation of an L 3 rather than an L~ phase is the opportunity to model an optimal curvau,re of the lipid monolayer. The stcucture of the L 3 phase can be seen as arising from a inched or disordered cubic structure. Such a disordering is favoured by weak interactions betwee,-, the hilayars. At higher iipid concentration and thus strong inrerbilayer forces, a cubic phase may form in equifibrium with the L 3 phase, it can be expected that I.~ phases also form with membrane lipids.

IV-B. Kinetics of the phase transitions Siegel [54-58] has presented a kinetic model of the L ~ / H n phase transitions, and the formation of cubic phases and isotrnpic structures (Fig. lb). In hi~ model the first structures that form during the L , / H . phase transition are inverted mieellar intermediates (IMIs). which form rapidly when two bilayers are apposed above the phase transition terllporatm¢ between ~ , to H n phases. It is found that approx. 10t°-10 It IMI structures per cm 2 of bilayer develops within milliseconds [54,57]. The shoat-lived I M l s then rapidly either aasemble into H n phase precursors via coalescence of IMls or form another type of a melastahle structure called interlamenar attachment (ILA). ILAs are bilayer interconnections between apposed lamcllae of the L~ phase and the structure of an ILA is very similar to the structure units building up bicontinuous cubic phases.

Thus. during the L ~ / H n phase transitions, there are two processes that compete for the available pool of IMIs. The rate PltA at which an IMI can form an ILA can be shown to be [54,57|:

pl~>ktn~o.l_ls

t

where k! is the coalescence re,to constant for the lMls and n~ is the steady-state number of lM[s per unit area of appesed bilayer/bilayer interfaces. Both n ° and PtLA affect the proportion of IMls that form ILAs. The value of n~ is determined by the ease with which bilayers can be closely apposed and by the radius of the water cylinders in the HIt phas~ at ,~quilibrium. PILA is determined primarily by the rehifivc mdgnitude of the lipid concentration fluctuation in each monolayer surface required fez the ILA formation, which in turn is determined mMnly by 2", the ra:;o of "he areas per lipid hea,:i group at t[~p lipid/water interface in the L~ and H u phases. Siegel showed that for Z ~ 1.2, PIL~ will be equal to 0,l - l s - t , i.e., approximately equal to ktn°. so that the formation of the ILAs and the H n precursors occurs at rates that are of the same order of magnitude. It can then be concluded that enough ILA,~ should form so that isotropie and cuhic structures could form. The cubic phase structures are formed by an accumulation of the *LAs in hi,.it :ancentration. Since the 1LAs hay ~. lifetimes that are ve.'y much longer than those of the IMIs. the ILAs will gradually accumulate ~ntil almost all the IMlayers have been converted into these structures. Note, however, that in this rather crude model the ILAs are not necessarily more stable than planar bilayers - they merely represent kinetic traps. The strong metastabillty of cubic phases, mentioned in the introduction, CaR thus be understood with this model. The ILA formation mrdces the L ~ / H . transition slower or more hysteretic, because the lipid is tied up in structures that cannot readily form either of ibese phases. Thus, the kinetics of the formation of bicontinuous cubic phases may he, and often are, dependent on the thermal history of the sample. It has been shown that if a system is subjected to beating/cooling cycle~ between temperatures just below and above the L , / H n phase transition temperature, more and more lipids forms ILAs (and eventually cubic structures) with each successive cycle. Therefore, these lipids are not able to caltibit the phase tr~:~ition between the L~ and H n phases [59]. Thus. again the curvature of the monolayer pLtys a dominant role also in the kinetics of the formation of cubic phases. Since metastability is easily obtained for cubic phases it ntight b~. ttifficul! to determi.ne the true phase diagram. This has recently been di*cussga by Siegel [57] and Grnner ct aL [18]. An example of the experimental difficulty that one faces has been dlustrated by Gruner ct aL [18] in Fig. 14, where they investigated the phase behaviour of di-

234 222 311

~oo

iii i*o

12111 ~zool

t

.o_

/.o~

"N o~. o

~.

Y

Xm3m

(11o1 12~o) 12111

Pno3mpn t ~ LlO III

lit 220 221 310

~.o-o Fig. 14. (a) A densitometerizationthrough an X-rayfilmexposure of a spe~m©nof DOPE-Me/wat~formin8 two cubic phasesin ~quilthli~. The tick marks are the expected positionsof the diffraction peaks from two cubic latticesthat are indexed (hk/) near the ticL The indices without parentheses are consistentwith either the Pn3 or Pn3nl space groups. The indicesenclosed in parentheses ale for an lm3m spacegr0ep; however, the ord~s obeyed for the second latti¢: do not unequivocallydetermine the space group. (b) S¢-attenn8 angics of the peaks of the two cubic latticesof (a) vs. ( ~ + k 2 + i2)1/2 for the hkl of the expectednon~ero reflections. All points from each latticeshould fall on a smtightline whose slope is used to determinethe dimensionsof the unll cell. From Ref. t 8.

oleoylphosphatidyI-N-m o n o m e t h y l e t h a n o l a m i u e (DOPE-Me). This system was found to show a complex nonequilibrium hehaviour. In the simple model by Siegel it was also questioned whether all cubic phases arc tiiermodynamicaliy stable phases, since the chemical potential of the lipids in the ILAs and the structure formed from them are almost identical to that of the Lo phase from ~]-~ch thcy form. However, the model is not ye, sufficienffy developed, which implies tha~ very small perturbations to the chemical potential of the ILAs might make the cubic phase thermodynamically stable. However, since the ILAs and the structures that form from :Item car, havc a lifalimc of hours, days or longer times, it may be very difficult to decide when the system has attained equilibrium. This is a ,.,:en-t,,~...~ exp.*'imental problem to the workers in the field but it has not been pointed out in the literature until recently [18,57]. V. Experimental methods to study cubic phases and isotrapic structures

V-A. X-ray diffraction Among the liquid crystalline phases of membrane lipids the structure of the cubic phases is undoubtedly the most difficult to determine accurately. The obvious method to be used for structural determinations of this kind should be X-ray diffraction, which also has been used since about 1960 on a large number of amphiphile systems formin 3 cubic phases. Unfortunately, space

group assignments are tentative but for a few cases. For no membrane lipid sample so far studied has there been a sufficient number of reflections for a completely unique identification. Fm'thermose. X-ray diffraction detects only structures with a long-range order, and therefore sample he~erugeneity cannot be excluded. From the X-ray diffraction patterns alone, it is sometimes even difficult to determine whether a cubic stnm. tare is primitive, body-centred or fare-centred. However, often it is possible to obtain ~nough information indicatin8 primitive or body-centred structures, for example from a study of the number of absent reflections for the cubic space groups based on an evaluation of reflections with l g h t + k 2 + l t < 6 8 [60I. It should, however, be meff,.ioned that a reflection may also be accidentally suppressed. Here, one possibility is the fortuitous coincidence that the structure factor is zero at the position of an order of reflection. This problem is often solved by y o u n g the water content and temperature, but this approach is seldom applicable to cubic phases which often exist over a very narrow concentration and temperature region of the phase diagram. In some cases problems also- arise if the cubic phase grows to large monocryst~s. On the other hand, if these munocrystals become very large it might be possible to work with monodomains. For one cubic phase, namely an anhydrous strontium soap at elevated temperatures. Lutzati and Spegt [20] unarabiguously determined the structure to belong to the body.centred space group la3d. The next step in the

235 determination of the phase structure will be to arrange the amphiphihc molecules in agzrcgamz in the cubic unit cell. For ]ameiiar and hexagonal phases this is a relatively simple matter hut not so for cubic phases where this difficulty is still a major problem. Luzzat~ and co-workers [61-63] in their early works first proposed a face-centred cubic structure to be formed by globtdar lipid micelles surrounded by water. Later they changed the model to consist of water spheres embedded in a hydrocarbon matrix. Not until 1967 Luzzati and Spegt [20] suggested a structure in which short rod-llke aggregates are linked three by three at each end forming two interwoven three-dimensional networks, i.e., the .qructure is bicontinuous. This cubic structure of la3d lymmetry was based on the early series ot papers by Wells [64] on the geometrical ha.~is of crystal chemistry, This structure is accepted by most workers in the field of surfactants, where the rod-like unit is built up of amr, hiphiles. However, for membrane lipids forming la3d structures, these are usually of the type of reversed cubic phases, and recently other proposals of the structure have been suggested [28,39,41] (subsection Ill-C). Fig. 14 illustrates typical X-ray data from two cubic phases formed by DOPE-Me and water. Here a deitsitumctrlzation of an X-ray film exposure and the scattering angles of the peaks of two cubic lattices vs. ( h 2 + k 2 + 1 2 ) 1/2 are shown. The peaks ~ o w n in Fig. 14a index as two lattices (Fig. 14b). One of the lattices consists of nine orders of diffraction and the space group cuns~..,tent with this resdit is Pn3m or Pn3. A second lattice giving three orders was also observed. A definite latdce assignment on so few orders is not po~ible, but a body-centred space group was suggested

I181. A final comment zngarding the X-ray method is that it might be possible in the future to improve the technique further, foe example at the synchrotron facilities that are now avails.hie at several places. Perhaps it may then be possthle to get among other things an appropriate wavelength dependance of the diffraction diagram. In a very recent developmtmt an at':empt was made to utilize the information available in the diffraction intensities by using a new algorithm [29]. However, at presemt X-ray diffracfio~t has still to be complemented with other methods, i~ particular N M R (subsection V-D), in order to get an acceptable determination of the structure of cubic phr~se-s. V-B. Tlrae.resolved X.ray diffraction Recently, X-ray diffraction has been utilized for studies of the stracture dynamics and mechanisms of phase transformations, A time-resolved X-ray diffraction method, using synchrotron light and a two-dimensional live-time X-ray imaging device, has been developed by Caffru~ [65,66] for studies of h~nsitions invniv-

ing me L a. ~uoic, H~l and fluid isotropic phases. S~veral lipid/water systems have been investigated. From eaeasurements on the transitions of L J H n phases for dihexadecyI-PE at hydration levels ramglng from O to 60% (w/w) water, it was found that the transitions were: (l) repeatable; (2) reversible; and (3) had an upper bound on the time required to complete the transition (transit time) that was 6 3 s. Similar results were obtained for the transitions between different cubic phases in two monoacylglyeerol/water systems [66]. Here, in some cases pronounced hysteresis effects were observed and very slow transit times ranging from 0.5 to 30 rain were found. In this work, also structural parameters and thermal expansivity of the long spacings for the various phases as a function of salt (NaCl) conLer,~'ation were presented. Recently, Oruner [67] published a detailed 1~;5~-",, m,'fic,'e on the time-resolved X-ray diffraction method for investigations of biological systems. V-C. Elec ron microscopy Electron microscopy (EM) methods have long been utilized for studies of lyotroplc liquid crystalline phases. The technique where electron micrographs are obtained from replicas after freeee-fracturing has proven to be very suitable for studies of the aggregate structures and phase structor~ formed by membrane lipid/wates mix.. tures (for a review see Ref. 68). Liposomes, and L~ and HII phases give rise to characteristic patterns on electron micrographs with thi~ technique. About 10 years ago a new type of structure, "lipidic particles ) (LIPs), was observed in [ipid/water mixtures [69]. The formation of LIPS ha~ attracted much attention. ~ c e these structures tlave been proposed to be involved in processes such as membrane fusion and transport of proteins, i;pids and polar solutes across membranes [70]. It has been shown that the_ LiPs eza'~ v,at9 in size (6-28 nm in di:anetes) (Ref. 68 and Siegel, D., Bums, J . L . Chestnut, M,H. and Talmon, Y., unpublished data), in shape (e.g,. semispherical pr0trt, sions and volcano-like protrus:ons with or without depressions in the centre), and they can be organized in several ways: ,andomly distributed, or arranged in row~, .~n a bilayer surface, and two- or three-dimeosional regular arrays of LIPs [68]. The molecular organizati.3n of the lipids in the LIPs has been extens*vely discussed, partly due to these variations in their appearance. Fig, 15 illustrates some frecze-fmctnre electron mictofp'aphs o* different lipid/warer mixtures giving rise to threc-dimeasiunal arrays of LIPs, Such sro.w.*.ures have bean interpreted to represe.nt a eul;!c phase or a quasi-crystalline strantnre ballt-np of t:osed lipid aggregates of the reversed type. i.e., strtlctares with discontinuous wares regions (reversed mice !lea) a-el with continuous hydrocarbon regions [68,71-7. j. R e ~ . t l y , it

236

Fig. 15. Free,e-fracture electron micrographs of different lipid/water raixtures forming cubic plmses. (a) DLiPE/eg8 PC (molar ratio 85/15) dispersed in buffer containing NaCI and histidine; quenched from 22° C after being heated to 40t'C; from Ref. 181. (b) DLiPE/POPC (molar rs:io 95/5) dispersed in buffer containing NaCI, EDTA and histidine; quenched from 20 o C; from ReL 73. (e) DPG/dibucnine (molar ratio 1/1.5) dispersed in buffer containing NaCI and EDTA; quenched from 0oC; from Ref. 72. (d) MGaiDG/DGaiDG (2:1, w/w) dispersed in water containhi8 NaC1; quenched from 50 o C; from Ref. 97. (e) DOMGhiDG/DODGhiDG (mohir ratio 3/1) disper sod in heavy water; quenched from 50°C; by courtesy of Dr. A. Verkleij. (D DOMGluDG/2H20 Wilh 9 wt¢~ 2H20; quenched from 20°C~ from Ref. lI. (g) Sunflower oil monoacylg]ycero]s/cytochrome c/water; from Ref. 10S.(h) E88 LPC/water; from ReL 105. White bars represent 100 m .

237 was shown (Ref. 30 and references therein) by X-ray diffractior, and the N M R pulsed field gradient technique that all the above-memioned systems form cubic pha~es which with one exception are bicontinuous (subsection III-B). The only exception known hitherto is the cubic fiquid crystalline phase formed by some saturated LPCs at high water contents (t-=g. 7~ ~her~ a discontinuous aggregate structure of the normal type is present [23]. The similarity in the general appearance of all these pictures is striking; Fig. 15a-g are obtained from bicontinuous phases while Fig. 15h is obtained from tlie phase with closed aggregates. Thus, it does not seem to be possible to distinguish between ~he two fundamentally different cubic phase structures with this EM technique. Randomly distributed LIPs, and rows of LIPs, occurring on a brayer surface have usually been interpreted to be different intermediate structures formed during membrane fusion (see Fig. 6 in Ref. 68): (1) volcano-like protrusions without dcpressians in the centre may rellect a preftlsion stage where two ne/ghbouring oilayers form :, contact point ('adhesion stage'); (2) well-defined semi-spherical protrusions may rep~sent reversed micelles within a bilayer ('joining stage'): and (3) volcano-like protrusions with depr.~,siot=q in the centre may reflect a contact-structure obtained after th= fusioh of two membranes ('fission stage'). The structural interpretation of LiPs has done considerable progress through the development of a new EM technique, limeresolved cryo-TEM with resolution in the time-regime of seconds (Siegel, D. et a l , unpublished data), and the development of a theory describing the formation of intermediate structures during she transformation between L= and H H phases [54-58] (subsection IV-B). Briefly, the theory predicts that short-fired IMIs (conesponding to reversed mlcelles within a bilayer) and long-lived ILAs (corresponding to the contact-structure betweea fused membranes) are formed (compare Fig. 16 with Fig. 6 in Ref. 68). The results obtained so far with time-resolved cryc-TEM can be summarized as follows (Siegel, D. ct at., unpublished data): (1) the ILAs can be visua!ieed direedy, and the ILAs appear to correspond to the LIPs observed by freeee-fractare EM; this suggestion is supported by the observation made by Verldeij and colleagues that the LIPs seem to be stable structures [68]; (2) the ILA is an intermediate structure in membrane fusion; (3) the 1LAs mediate the assembly of reversed cubic (12) phases, Verkleij and colleagues have probably observed ILAs both with the freeze-fracture EM technique (volcano-like protrusions with depressions in the centre) and a thin-section EM technique (joining points between bilayers in a 'honeycomb' structure) [74], An advantage with the two EM methods described above is that several aSgregate a n d phase structur¢~ can easily be observed on the same micro-

graph.

L a phase

Ha phase precursors ILA (fusion)

(leakage)

® Isotropic or

~u ~h~se

Invert~.d Cubic P~ases

Fig. 16. Schemallc according to Siegel [55-571 of the fusion and d~tabilization (Ia i phase fotanation) pathways for hposomescomposed of polymorpld©llpid.g.Fl~m ReL 131. Reecndy, Talmon showed by TEM [75] that some surfactant and lipid micellar solutions can form cubic structures at the circumferenoe of an EM grid. Electron micrographs of micellar solutions of PaLPC and egg LPC are shown in Fig. 17. it was also determined by small angle X-ray scattering that these lipids form cubic phases composed of globular micellar aggregates (cf. Fig. g and subsection V-D). V-D. Nuclear magnetic resonance V-D.L Diffusion measurements

It is obvious that the complex structure of the cubic phases cannot be solved by only one physical method. As mentioned above X-ray diffraction alone will net be able to give a conclusion answer and we have to look for other methods to get further complementary information. N M R techniques have shown to be particularly powerful for this purpose, above all the N M R pulsed field gradient method, for the determination of translational difhsion coefficients of both the fipid and water components. A larger number of cubic phases forv~ed by membrane lipids have been investigated with this method (Refs. 11-13, 23, 26, 30, 76, 77 and 146-148; and Eriksson. P.-O. and Lindblom, G., unpublished data). In general the N M R spectra of cubic phases exhibit narrow high resolved peaks (for an exception see below on lysolipids). This is due m the fact that the molecular motion is isotropic and all the nonscalar interactions are averaged out. The N M R signals of cubic phases are therefore similar to those of a mieellar solution and are consequently not sensitive to details of the structures of

23g

Fig. 17. Lowtemperaturetransmissionelectror~microgtaphof a sample¢orlsistingof l ~ l c ml~llar solutions(25 ,wt~$)o[ (it) PaLIg2and (b) egg LPC. This mi~ograph suggeststhe formation of a cubic p h ~ structure which was also confirmed by small ~ X-rayscattering.For technical details,scc Rd. 7S. By~unesy of Dr. Y. Talmon. isotropic phases. On the other hand, a prerequisite for the conventional N M R diffusion method [78] to be used is the occurrence of narrow peaks in tile N M R spectrum. The main advantage of this method is that the diffusion coefficient can be measured directly without using probe molecules and no model dependent assumptions have to be made~ A schematic picture of the experimental set up is shown in Fig. 18a. The two radio-froquc~acy pulses, 90 ° and 180 °, give rise to an echo, the height of winch is dependent on the molecular diffusion. The pulsed magnetic field gradients (Fig. 18a) can be varied and set very accurately and define the time during which diffusion is measured. For membrane lipid/water systems, the chosen diffusion times correspond to translational diffusion distances of approx. 1 and 10 pm for lipids and water, respectively. Thus, with this N M R method the translational motion is measured over distances that axe much larger than the dimension

of a single micellar aggregate. This means that restricted diffusion, for example a lipid molecule in a micelle or a water molecule in a reversed micelle, can be conveniently detected, since the distance of translational diffusion of the captured molecules in the mieelles will be too small to have an effect on the measured diffusion coefficient, although the molecules can move fast within the micelles or the cavities. This is illustrated in Fig. 18d. Note that Fig. 18c and e show two cases (lamellar and cubic structures) where the translational motion can occur over macroscopical distances. It is then obvious that, from a comparison of the measured lipid translational diffusion coefficients in the cubic and lamellar liquid crystalline phases, it is possible to differentiate between the two fundamentally different types of cubic phase, namely the bicontinuous ones and those built up of closed micellar aggregates. To be able to manage tins, the lipid diffusion coefficient in the lamel-

239 a

b

Ll\n !I a ¢

NN d

e

Fig. I8. (a) Schematic pict~ of the NMR diff~ion method wilh 90-r-tg0 ff pulses and pulsed magneticfield gradients~ 8 is the width and ~t the spa~n8 between the field gradient pul~. F1D, free indueti~ d~ay, (b) lU~trationof the oli~taaon of the ~oseopieally aligned I~ell~ ~ p l c ~l~tive to the exte~al magneti~field, Be, and Ihe matgaeacfield ~adi~t, g. (c-e) Illustration of the lipid tr~slational diffusi~ in lipid/water p h ~ of ditfe~nt structures during a time of milliseconds. (c) Lamellarphase; the lipids move freely in Ibe aggregateplane and are able to transverse over large distances,(d) A hypotheticalcubicphase built up of reversed mieell~; the lipid5 move freely within the aggregate-~,whereas intera&~,regate motion is hindered by hydrophobia interaction,re) nlconanuous cubic ph~e; the lipids move freelyalong the lamellaragglegates,whichare connected to each other. Adapted from Rats. 30 and 79.

lar phase, the so-called lateral diffusion coefficient, has to be measured. Unfortunately, the standard N M R techniques cannot be used in a straight-forward way to obtain this coefficient. Due to the presence of static dipolar couplings the effective relaxation time (T2) is too short for such measurements to be leasable. However, this problem can be circumvented by aligning the lamellar phase maeroscopically and by orienting the sample in the magnetic field at the so-called magic angle, 54.7 ° , so that the static dipolar couplings vanish (Fig. 18b). With such an oriented sample the convert-

tionai N M R method can be used for det~a'mina!~on~ of lateral diffusion cnefficiems [79]. It has been found for a larvae number of lipid systems that in bicontinuous cubic phases the measured lipid diffusion coefficient is of the same order of magnitude as that obtained for the corresponding lamellar phase [26,30] (Fig. 11), while for cubic phases composed of closed aggregates, the measured diffusion coefficient is between one and two orders of magnitude lower than for the corresponding lamellar phase [13,79]. The hieontinuous cubic phases determined in this way are marked with an asterisk in Table I. For the values of diffusion coefficients the reader is referred to the original references (see also Ref. 313 where many diffusion coefficients have been tabulated). Note that for the lamellar phase the lateral diffusion is measured direcdy, i.e., the translational diffusion along the plane of the lamenae is observed, while for the bicontinuous cubic phases the measured diffusion coefficient is an avera,~e of the local diffusional motion in the aggregates o,er the cubic structure. This property of the measured diffusion coefficients provides another possibility to gain further information about the apparent cubic strut.tufa. It is assumed that the local lipid diffusion coefficient is independent of the shape of the aggregate, i.e., the local diffusion coefficient is assumed to be equal to the measured lateral diffusion coefficient, whether it is a lameliae or a rod. This leads to the conclusion that the magnitude of the measured diffusion coefficient of the cubit" phase will he appreciably influenced by the shape of the aggregates building up the phase. Thus for rod-like aggregates the local diffusion coefficient can be determined to be three limes the measured coefficient of the cubic phase, while for lamellar aggregates it will be 3/2-times the measured diffusion eeefficient [26,79]. Fig. l l a and b show experimental results for two lipid systems, where the lamellar and cubic phases at constant composition transform into each other by a change in temperature. It can be inferred from this figure that in both systems the translational diffnsic, n in the cubic phase is best described by a motion in bilayer units 126,30]. Also the water diffusion has been studied for some cubic liquid crystalline phases 113,30]. As expected a reduction of the water diffusion coefficient, as compared to pure water (a factor of 3-5 depending on the system and the temperature), is observed. Fig. 19 shows an example [30] of a stacked-plot representation of proton N M R spectra obtained from a Fourier transform of the second half of the spin echo in an N M R diffusion experiment [80,81]. The peak on the left-hand side in the N M R speetrttm representing water decreases rapidly due to its fast translational diffusion. On the contrary, the lipids diffuse approximately three orders of magnitude slower thm~ water and therefore give rise to peaks of nearly constant heights (on the right hand side in the spectrum) in this experiment. The time scale

240 TABLE l ~pid/waler sysle~ f~ing

cublc p h ~ e s z ~ c t ~ e s

Lipid MO MO PaLPC OILPC Egg PC DOPC DLiPC Egg PC/sodium chelate 22-35 wt~ sodium chelate Egg PC/DG ~ 75-80 wt~ IX} Egg PC/DLIPE S5 wt~ DLiPE DOPC/DOPE 0-50 wt~ DOPE POPC/DLiPE 85-90 w[% DLiPE PE d PE ~ DDPE DOPE DOPE-Me DPG r/dibacaine 29 wt~ dibucalne Sodium sulfatide t DOMGluDG h DOMGIuDO/DODGluDG h 51 WI~ DODGIuDG MGalDG/DGalDG J 31 wt~ DGaIDG MGalDG/DGalDG t 34-50 wt~ DCaIDG GDNT I GL I Polar lipids Irom A. [md~awii Polar lipids from $. solfataricus

Watea-~nlent (wt~) 12-33 35-40 40 46 20-25 0-4 2-11 4 22-26 excess ~s 10 29-41 35--50 8-26 10-16 > 33 50

67 41 40-70 7-]5 10 10-20 3-15 7-13 0-It 18 20 40

Temperature ( o C) " 20 20 25 25 75 69 55 22 l0 40 70 7 4~ 58 75 llS 4

25 7 20 0 25 60 10 t5 6O 65 85 85

Proposed space group b la3d " P,,3/Pn3m * P43n/Pm3n la3d

P

P la3d * Pn3m Pn3/Pn3m Pn3/Pn3m ( + Im3m?l

p * P Ia3d * Ia3d * la3d la3d * la3d la3d gg3d PnJm

Rcf. 28 27 10.7.3 10 183 12,92 17 180 94 1S1 109 30 184 139 182 59 18 30 96 11 11,146 185 9

98 99

1l 98

" Low~t temperature for the formation of the cubic phase. b . denotes that the cubic phase has been determined to be bicontinuous by the NMR diffusion technique (see sub.:orion V-D.I). Diaeylglycerol derived from egg PC. [~laled from P. r e g i s . [~lalcd from B+ mcgaterium. Isotaled from bovine h~t.

s Isolated h I~latcd ) Isolated k Isolated I It~lated

from human brain. from A. ImdlawiL from maize chloroplasts. from wheat chloropl~ls, from S, solfa:a~icus.

in Fig. 19 c a n be considered as a n effective t i m e d u r i n g w h i c h the diffusion occurs. I t should also be m e n t i o n e d here t h a t it has even been possible to estimate the size of the unit cell of the bicontlnaous cubic phase f r o m a c o m b i n a t i o n of measurements of different N M R parameters [82]. So far this h a s been d o n e for simple surfactant systems, b u t there is n o reason w h y it should not be applicable to mareb r i n e lipid systems. Thus, by determining: (1) the relaxation times, T l a n d ?'2; (2) a q u a d r u p n l e splitting from which the o r d e r p a r a m e t e r of the lipid c a n h e obtained; a n d (3) the lipid translational diffusion coefficient, a n d by using a m o d e l in which the molecular motiolt occurs o n a sphere inscribed in the cubic unit cell, the radius of this sphere c a n b e calculated. T h e value of this radius should then correspond t o h a l f t h e

d i m e n s i o n of t h e u n i t cell. T h e structural p a r a m e t e r so d e t e r m i n e d b y N M R is in surprisingly g o o d a g r e e m e n t w i t h the X - r a y d a t a [82]. A similar investigation h a s also been p e r f o r m e d w i t h eleclron spin r e s o n a n c e ( W i k a n d e r , G . Eriksson, P.-O., L i n d b l o m , G., Kxistoffecsson, J., Burnell, E. a n d Stael y o n Holstein, J., u n p u b l i s h e d data). V.D.2. Bandshape

studies

Usually the N M R spectra o f optically isotrupic cubic liquid crystalline phases consist of isotropic L o r e n t z i a n lines, d u e to a fast isotropic molecular m o t i o n . H o w ever, for the cubic phases formed at h i g h w a t e r c o n t e n t s w i t h either l - l a u r o y l - L P C . 1-myristoyl-LPC or P a L P C (for p h a s e diagrams see Fig. 2) the spectral lineshapes showed obvious deviations f r o m a n isotropic Lorenggian

2.~1

l la -

-

9 e

----7 e

f

t

4 3

_

_

1ms

m plm Fig. 19. NMR diffusion experiment on a cubic phase with a molar ratio POPC/DLiPE 10:90 and with 37.3 wtg~ water. A stacked-plot representation of proton NMR spect~ obteined at ?°C from a Fourier transform of the second half of the spin echo. The limesshown in the figure can he seen as ~ eff~tivc fimcduring which tr~shitiunal diffusion ~urs. The peak on the left-h~d side in the sputum ~pr~nts the water, and the peak on the right-hand sideoriginatesfrom Ihe lipids. From ReL30.

form caused by a not fully averaged anisotropic interaction [23]. The 2H-, z4N- and 31p-NMR spectra (Fig. 20) appear to be a snperposition of two signals, a narrow one (isotropic motion) and a broad one (partially averaged anisotropic motion). However, the cubic phase samples were optically isotropic between crossed polarize~ Furthermore, changes in the water concentration or the temperature had no significant influence on the line shape or the relative intensity of the narrow and broad components. What kind of structure of the cubic phase is compatible with these unusual N M R line shapes? Since the cubic phase is located between the L t micellar solution and the H t phase in the phase diagram it can be expected to be composed of dosed micellar aggregates. which was also shown to be the case by N M R diffusion measurements [13,23]. Since two components were observed in the N M R spectra it is reasonable to assume that the phase structure is composed of two categories of aggregates, in one of which the static interactions are fully averaged by fast isotrupic motions, and in one of which any isotropic motion is too slow to average all static contributions. A structure [83] that fulfils the requirements concerning all the N M R data obtained is illustrated by the model in Fig. 8. In this structure short rod-shaped agsregates with an axial ratio of approx. 2

occupy the different positions in the cubic unit cell. It sho,#s that the micelles in the comers and in the centre of the cell are able, by a concerted motion of adjacent micelles~ to take on all orientations in the cell (isotropically disordered), while the mice-lies at the surfaces are hindered to rotate around one of the short axis (anisetropically disordered). With this model of the cubic structure the complex N M R line shape can be fully explained [23]. Recent attempts hy I.uzzati and coworkers [29[ to justify their old structural model [22], consisting of micelles located in cages built up by rod-like aggregates, are not successful, since the structure is not compatible with either the N M R diffusion or the N M R linewidth data. Furthermore, a recent determination by time-resolved fluorescence spectroscopy of the lipid aggzegation number in micellar and cubic phases of LPCs gave a value of shoat 100 (Lindblom, G., Johausson, L.B.-A., Arvidson, (3. and E~ik~on, P.-O., unpublished data), clearly indicating that this cubic phase consists of only closed aggregates. An attempt to understand the conditions under which these LPCs are involved in the formation of ;.he cubic phase consisting of micellar aggregates has also been undertaken [13]. From N M R diffusion and relaxation studies, information about the structure of the micelles of LPCs with different acyl chain length and unsatura-

242

~H

/\

'H

I

/

\

II~

/l

2s "c

S kHz

1 hHz

B

"N

"N

[

y ~;7¢.

£

~

kH~'~

O

P

[

,s.:

"...A

' 1 kHzt

P

S ppm

Bo

F

5 ppm

Bo

Fig 20. 2 H-, 14 N-and 31 P-NMRspeetraofliquidcwstalllnesamp.esof[2.2. ZH2lPaLPC and water, lA, CandE)Pureett b icphaee, tg, D andF) Mixtures of cubic and H I phases. 'l]lis figureilhistrat~ the unique NMR bandshapes obtained for this cubic phase, compatiblewith the strecture shownin Fig. 8. From Rcf. 23.

formation of the liquid crystalline phases, was obtained. It was found that for the LPCs forming a cubic phase between the L 1 micellar solution a"d the H t phase, the micelles remained small and globular over the whole micellar phase region (exceptionally, 1-stearoyI-LPC did not form an I= phase), while the micclles of 1-olcoyl-LPC (O1LPC) or 1-iinoteoyl-LPC are large and polydispersed. It seems then reasonable to assume that the low concentration limit of the cubic phase area is determined by packing COnstraints of the mieelles in the ~:uhlC structure. From the concentration dependence of the LPC micellar diffusion coefficients, and also from

the water diffusion coefficient in the cubic phase, it was found that the effective volume fraction of mieellar aggregates at the formation of the It phase is about 0.68-0.71 [13], i.e., the micelles are proximately packed (the volume fraction of close-pecked hard spheres is equal to 0.74). A micellar solution consisting of long rod=like aggregates, on the other hand, transformed directly to an H i phase at increasing lipid concentration. It is interesting to note that the formation of the 1: phase, as expected, is vet2, sensitive to small changes in the shape of the mlcellar aggregates. Thus. sohibilization of small amounts of oleic acid in PaLPC mieall~

243 is well known that surfactant mice]les grow upon solubilization of long-chained alcohel~ or fatty acids [5]. V-E. Other spectroscopic r~ethod~"

So far only a few other spectroscopic methods have bucn used to investigate the physicochemical properties of cubic liquid crystalline phases. There are, however. some investigations that can be briefly mentioned here. Concenting the shape of the aggregates building up the cubic phase it has been st)ggested that time-resolved fluorescence should be suitable [,°4[. Yhis idea is based on the measurements of order parameters of fluorescent probes at long decay times. The drawback with this method is the difficulty to find appropriate probe molecults. Time,resolved fluorescence can also be used to measure the aggregation number ot micel!e~ forming a cubic I~ phase [85]. Recently, a[~o electron spin resonance was utilized to determine the size of micellar aggregates in a cubic phase (Wikander, G., Eriksson. P.-O., Lindblom, G., Kristoffcrsson, J., Burnell, E. and Stael yon Holstein, J., unpubfished data). To in~stigate the hydrogen bonding properties at the aggregate interface in various cubic phases, Fourier transform infrared spectroscopy investtgations have been initiated (Nils, son, A., Holmgren, A., Lindhhim, G. and Rilfors. L., unpublished data). Finally, it should be mentioned that in principle it should be possible to get information about whether a cubic phase is bicontinuous or not from a study of fluorescence recovery after photobleaching (FRAP), since this method is based on the fact that, to be observed, the fluorofores have to diffuse over a maeroscopical distance into an area of the sample which has been previously bleached (the idea behind this method is thus analogous to the N M R diffusion method, cL Fig, 18), Such studies have also been initiated (Magnusson. K.E, and Lindhlom. G.. unpubfished data).

VI. Memblmne lipld/water systems |orming cubic phases By now cubic phases have been found to be formed by single membrane lipids, by binary mixtures of membrane lipids, and by total polar lipids extracted from bacterial membranes (Tabl~ I). Monoacylglycerols do not occur in biological membranes but are of biological relevance and have therefore been included in this r~view. These amphiphiles play an important role in the food industry, and it has been suggested that a cubic phase of monoacylglycerols is an intermediate state in fat digestion (subsection VIII.D) [86[. Lutton [15] construeted phase diagrams of ten 1-monoacylglycerol/ water systems and found that a viscous isotropic (cubic) phase was formed with monomyristin, monopalmitin. mo~ostearin, monoarachidin, monoelaidin, monoolein

and mf,nolinolein. Particularly the M e / w a t e r system has been studied in detail later. Two cubic phases are formed by this system (Table 1; Fig. 4l: one phase belonging to the space group laJd at lower water contents and one phase belonging to the space groups Pn3 or Pn3m at higher water contents {see Section Ill). A cubic phase is formed also by 2-monoolein but at higher temperatures compared to l-monoolein [871. The cubic phase of M e / w a t e r is able to incorporate various water soluble, amphiphilic and hydrophobic molecules. Exampies of such molecules arc: DOPC [16] (Fig. 3). glycerol. cholesterol, sodium chelate [88], globular proteins with molecular weights between 14-150 kDa [89], tetramethylsilane, oleic acid, monostearin, OILPC, and the hydrophobic peptide gramicidin (Eriksson, P.-O., and Lindblom, C~,, unpublished data). Lysolipids can occur in a biological membrane as a result of the action of phospholipas¢ A.,. "lhe phase equilibria in six 1-acyI-LPC/water systems have heen studied (Fig. 2) [10,13]. 1-LauroyI-LPC, l-myristoyI-LPC and PaLPC form a cubic phase built up of closed aggregates (Fig. 8; subsection V-D) in the concentration interval between the isotropic L t solution and the H i phase; this cubic phase belongs to the space group P43n or Pm3n (Table I). A cubic phase is formed also by 1-smaroyI-LPC, OILPC. and l-linolcoyl-LPC, but this phase is formed in the concentration interval between the H t phase and the L , phase (Fig. 2l. From measurements of the lipid translational diffusion coefficient in the cubic phase of OlLPC/wuter it was concluded that this phase is bicontinuous [30]. Phosphatidylcholines (PC) are often regarded as typical examples of membrane lipids forming a bilayer structure and thus a lamellar phase; in excess water the lipid forms a lamellar phase up to at least 9 0 ° C with varying degrees of aayl chain unsaturation 17,90]. However, at low water contents and at high temperatures PCs are able to form nonlamellar phases [1,183]. Egg PC forms a cubic phase belonging to the space group la3d (Table I) and even the fully saturated DPPC forms a cubic phase at these low water concentrations between approx. 80 and 100°C [91]. A complete phase diagram has been constructed for the DOPC/water system [92] and it is seen that the cubic phase is formed at somewhat lower temperatures with the PCs containing only unsaturated acyl chains compared to DPPC and egg PC (Table I). Chloroform, diethyl ether, haiothane [24]. and n-dodecana [12] can be incorporated into the cubic phase obtained with the unsaturated PCs, and a cubic phase is formed also when DOPC is mixed with formamide, methylformamida or dimethylformamide instead el' water [92]. The unsaturated PCs can form a cubic phase together with sodium chelate (Fig. 6), a dlacylglycerol, and unsaturated phosphatidylethanolamines (PEs) (Table I). The translational diffusion coefficient has been

244 determined in the systems egg PC/sodium cholate/ water, dilauroyl-PC/sodium cholate/water [76], 1palmitoyl-2-oleoyI-PC (POPC)/sodium cholatc/water, and DOPC/sodium cholate/water [77] and it was concluded that the cubic phase is bicontinuous. Diocylg]ycerols are often formed ,n the membranes of eukaryotic cells in response to various extracelhilar signals [93] and the influence of these lipids on the phase equilibria in membrane lipid/water systems is therefore of inler~st. A cubic phase with a primitive symmetry is formed by egg PC and a diacylglycerol derived from egg PC at high contents of water and diacylglycerol (Table l). However, it is noteworthy that if the diacylglycerol is mixed with egg PE, an H . phase is formed at 37°C already with 7 wt% diacylglycerol [94]. Up to 50 wt% of dioleoyl-PE (DOPE) can be incorporated into the cubic phase of DOPC/water (Table I) and the D O P C / D O P E phase is bicontinuous [30]. A bicontinuous cubic phase is formed at low temperatures with POPC/dilinoleoylPE (DLiPE), and egg P C / D L i P E can form a cubic phase ia excess water (Table I). PE with unsaturated acyl chains are often taken as examples of membrane lipids able to form an Hit phase. However, four different PE species have been shown 1o form a cubic phase (Table I). Two of the PEs have been isolated from natural sources; the larvae of the black blow fly Phormia regina and the bacterium Bacillus megalerium. PE from P. regina forms a cubic phase with a primitive symmetry at pH 2 and at high water contents, while PE from B. megaterium forms a bicontinuous cubic phase at low water contents belonging to the space group la3d (Gulik-Krzywicki, T. and Rilfors, L., unpubiished data; Fig. 21). Two cubic phases can be formed with the short-chain ether analogue didodecyl-PE (Table I; Fig. 22). Both phases are formed at high temperatures, one phase at lower water contents and one phase that belongs to the space group P n i m at high water contents. DOPE/water mixtures containing

RHTQILIPID (real I m o l )

HII. H2O

B*Hll

100

.~)

"/ '///"

Lu.H20

L0..~0

zo ~ ' ' ~ I L ~ ' ~ 20

0

~_~ 40

' F,..~E56

w t % HZO

Fig, 22. Phasediagramof the systemDDPE/H 20. Q= and Qb cpbic phases. Other not~.lions accordingto ReL 182,and legends to Figs. 2 and 3. Solid lin~ correspond to boundaries of stable phases and

dashed lines to boundaries of metast,~blephases, which am denoted in parentheses, From R¢¢.182.

approx. 50 wt~ water form a cubic phase that belongs to the space group Pn3 or Pn3m (Table 1) if the sample is cycled between - 5 and 15°C hundreds of times. Before the cycling is started DOPE forms an L , phase at the lower temperature and an Him phase at the higher temperature [95]. DOPE-Me forms two cubic phases in equilibrium at high water contents (Table I, Fig. 14); the dominating phase belongs to the space group Pn3 or Pn3m while the other phase probably belongs to the space group Im3m. The cubic phases discussed hitherto are formed by nonionic or zwltterionic membrane lipids. Two exam-

60 Q

Let

5o

j

Lo 1~

E p-

LG*ZH20

so

5 10 15 5 10 2HzO/lipid (mol/moi) 2H20/lipid (mol/mol) Fig. 21. Tentativephase diagrams for the systemPE/waterwith PE i~lated frm B. mesaterium. (a) PE isolatedfrom cells grown at 20°C and having a molar ratio iso/anteiso acylchains of 0.3. Ib) PE isolated from ~lls grown at 55°C and having a ratio iso/anteiso acylchains of 3.2. Notations according1olegendsof Fig.2. Data tak,~ from Rer. | 39+

24_~ pies are known where anionic lipids participate in the formation of a cubic phase (Table l). If diphosphatidylglycerol (DPG) is mixed with the local anaesthetic dibucaine in a molar ratio dibucmne/OPG of 1.5 a bicontinuous cubic phase with primitive symmetry is formed with 41 wt% water. A glycosphingolipid with a sulfate group (sodium sulfatidc) forms a cubic phase with primitive symmetry. This phase has the same location in the phase diagram as PaLPC, i.e., in the concentration imerval between the L I micellar solution and the H I phase [96]. Glycerolipi0s with polar head groups consisting of one or two sugar molecules occur in tbe membranes of both prokaryotes and eukaryotes. MGluDG and digiucosyldiacylglycerol (DGHIDG) account for 55-75 tool% of the membrane lipids of the bacterium. A. laidlawii [47]. Dioleoyl-MGhiDG (DOMGHIDG) forms a bicontinuous cubic phase at low water contents which belongs to the space group laid. Mixtures of DOMGluEN3 and dioleoyl-DGhiDG (DODGHIDG) can ,also form a cubic phase belonging to this space group (Table l; Seddon, J.M., Wieslander, A. and Lindblom, G,, unpublished data). A cubic phase consisting of D O M G l u D G and D O D G I u D G exists in the concentration intervals 30-60 wt% D O D G l u D G and 6 wt% to excess water [11]. Finally, :he total polar membrane lipids from A. laidlawii can form a cubic phase (Table l). Approx. 75-80 tool% of the polar lipids in the chloroplast membrane of Ligher plants consist of MOalDt.i and DGaIDG. In c,'~*trast to DOM(31uDG, MGalDG from wheat chloroplasts does not form a cubic phase, probably because of the greater degree of acyl chain unsatutation in the galactolipid [9]. Mixtures of MGaiDG and D G a l D G can, however, form a cubic phase belonging to the space group la3d (Table I). The phase formed by the wheat chloroplast gaiaetolipids has been shown to be bicontinuous Mixtures of MGalDG and D G a l D G most probably form a cubic phase also in excess water since fi%'mze-fracture electron mierographs exhibiting three-dimensional regular arrays of U P s (subsection V-C) have been obtained under such conditions [71,97]. The phase equilibria of dtttesent membrane lipids and membrane lipid fractions isolated from the thermoacidophilic amhaebaeterium Sulfolobus solfmaricus have been studied [98,99]. All the polar lipids of this organism are derived from the two compounds glyceroldialkylglycerol telraether and giyceroldialkylnonitol tetraether (GDNT) which consist of two biphytanyl chains etherlinked to two glycerol molecules, and to one glycerol molecule and one nonitol molecule, respectively. A variety of polar head groups are linked to the glycerol and nonitol molecules, but 20-30% of the glycerol molecules in a total fipid extract are mlsubstituted [98,99]. G D N T forms a cubic phase at low water cow tents that belongs to the space group la3d (Table 1).

The biological relevance of this obsctwation is. however. questionable since all the polar head groups had been removed by hydrolysis. The phase equilibria were also studied in mixtures of water and the total polar membrane lipids from S. solfatar~cus: the polar head groups wert= left intact in this case. Two cubic phases were formed at high temperatures: one phase belonging to the space group la3d at lower water contents and one phase belonging to the space group Pnim at higher water contents (Table I). Fir.ally, di:fere,tt fractio~ls of the polar lipids were investiga|~,~l, A glycolipid fraction (GL). consisting of 70% G D N T with/]-o-glucopyrano:;e linked to the nonitol molecule and 30% giyccroldialkylglycerol tetraether with fl-D-gaiaetopyranosyl-fl-L'galactopyrannse linked to one of the glycerol groups, forms a cubic phase belonging to the spac~ group l a i d at low water contents and high temperatures (Table IL A remarkable conclusion was made concerning the structure of the vario~as liquid crystalline phases built up by the dialkyl tetraether lipids carrying one unsubslituted glycerol molecule. These polar groups are asserted to he located within the hydrocarbon region, and the water/hydrocarbon interfaces are occupied preferentially by the substitaled glycerol and nonitol groups. The nonlamellar phases are formed only by lipids or lipid mixtures containing unsubstituted glycerol groups [98.99]. In conclusion, membrane lipid/water systems have been found to form cubic liquid crystalline phases under a variety of conditions: at temperatures ranging from 0-150°(;, and at water contents ranging from the anhydrous state to excess water. With two exceptions. cubic phases have so far been observed with nonionic and zwitterionie membrane lipids, but experimental results indicate that anionic lipids can participate in the formation of i~otr~p~c ~tw~otur°~ whe; mi~?d "~;:~ lipid forming an Ht] phase (Section VII). An interesting observation can be made when inspecting Table h the cubic phases belonging to the space group lo3d are formed at water concentrations below approx. 35 wt%, and the cubic phases belonging to the space groups Pn3 or Pn3m are formed at water concentrations above 35 wt%, irrespective of the membrane lipid involved (subsection Ill-C). More experiments have to be performed in order to confirm the general validity of this observation. Vii. Membrane Ilpld/watm' systems giving 3Ip-NMR speotra with a narrow syntmmtrical signal An extensive series of papers have been published by Culhs` De Kruijff, Veskleij and colleagues in which they have studied the phase equilibria in various membrane lipid/water mixtures with ~tP-NMR [701, A powder sample of anisotropic liquid crystalline phases, such as the lamellar and hexagonal phases, give 3tp-NMR spec-

246 tra with charzcteristic line shapes [100,101]. It is generally found that 3 iamellar phase gives rise to a spectrunJ with a high-field peak a n d a l o w - f i d d ~houlder, i.e., a negative chemical shift anisotropy. A hexagonal phase generally gc,aermes a spectrum with a Iow-fic;d peak a n d a high-field shoulder, i.e., a positive chemical shift anisotropy, where the m a g n i t u d e of the chemical shift anisotropy is reduced by a factor of a b o u t 2 [102] as c o m p a r e d to the spectrum from a lamellar phase formed by the same l i p i d / w a t e r system. However, Cullis, De Kruijff, Verkleij a n d colleagues often also o b t a i n e d a n a r r o w symmetrical signal in the 31P-NMR spemra. Such a signal arises from lipid struelures allowing isotropic motional averaging, for example, an L I or L : micellar solution, small vesicles, or a cubic liquid crystalline phase, W h e n the N M R spectra are recorded from l i p i d / w a t e r mixtures w i t h very high water c o n tents there are considerable difficulties to m a k e a distinction between the different structural alternatives. If a l i p i d / w a t e r system forms a m i x t m ~ of phases with no, or slow, exchange of lipid molecules, o n the N M R time scale, between the different phases, a n N M R spectrum with a superposition of signals o r i g i n a t i n g from these phases is obtained. O n the o t h e r h a n d , a rapid exchange of lipid molecules between a n isotropie a n d a n anisotropic phase results in a reduction of the static parameter, in this case the chemical shift anisotropy; a n a r r o w symmetrical signal is not observed in the N M R spectrum, Thus, only a 3 t p - N M R signal characteristic for a lamellar phase is expected to be o b t a i n e d if pits a n d bulges are present in a lipid bilayer (Fig. 4 in Ref. 103), since the exchange of lipid molecules between the different lipid structures is p r o b a b l y rapid. Moreover, a reversed micelle within the bilayer (Fig. 4A in Ref. 103) is situated in a n anisotropic s u r r o u n d i n g which a priori n'.7=:i= : h a t a n a r r o w symmetrical N M R signal should n o t be obtained. A parallel c a n be d r a w n to the situatiol~ where a N a + present in a lamellar phase gives rise to a q u a d r u p o l e splitting 1104] in spite of t h e spherical nature of the ion. Consequently, a discrepancy exists in some publications w h e l e a certain l i p i d / w a t e r system h a s been studied with 3 1 P - N M R as well as w i t h freeze-fracture E M . T h e N M R spectra exhibit a n a r r o w symmetrical signal whereas the micrographs show extended bilayers with L I P s arranged r a n d o m l y or in strings [103], instead of three-dimensional arrays of L I P s (Fig. 15). T h e isotropie phase detected by N M R h a d probably broken d o w n d u r i n g the freezing procedure a n d it is likely t h a t intermediate lipid structures are seen o n the micrographs [68]. Gulik-Krzywicki et al. [105] have pointed out t h a t w h e n the w a t e r c o n t e n t of a phase exceeds 30 w1%, only ultrarapid cryofixation m e t h o d s conserve, with varying degrees of success, the initial sample structure; a highly ordered phase, like a cubic phase, m a y be b r o k e n d o w n into a n a m o r p h o u s like material. T h e assumption that the L I P s represent

intermediate lipid structures is also s t r e n g t h e n e d by recent studies, ,sii,g fi~ne-reselved cry~-TEivi, nl ,~hiuil intermediate struelures were created u n d e r controlled conditions (Siegel, D. et al., u n p u b l i s h e d data) (subsection V-C). I n spite of the difficulties to give a structural interpretation of n a r r o w symmetrical J I P - N M R signals, a s u m m a r y of m e m b r a n e l i p i d / w a t e r mixtures giving rise to such a signal is presented in T a b l e 11. I t should be n o t e d that several of the systems collected in this table give rise to 3 t P - N M R spectra representing L , a n d H n phases in .~ddition to the isotropic structures. I t is seen in Table 1 that PC species c o n t a i n i n g m o n a - a n d / o r d i - c i s - u n s a t u r a t e d acyl chains f o r m a cubic p h a s e at low TABLE II L~pid/water systetns giving N M R spectra with a spectral c o m m e n t arising from lipid st~ctu~s allawlag i~troplc morzanal averaging

Lipid

Didocosahc^aenoyl-PC DLiPC/chol~terol 50 ~1~* chol~t~ol DPPC/MO 67 molto MO Egg PC/MGIuDG b 50 tool% MGIuDG Egg PC/soya PE 85 mol~ soya PE DOPC/DOPE/cholesterol 25 real% DOPE 50 mol~oenolesterot DLIPt:/DEPE 30-80 mol% DEPE DPPE/MO 67 real% MO DPG ~/Ca 2. Ca 2÷/DPG = 0.7 (real/too0 DPG g/~ya PE/Ca 2÷ 67 mol~ soya PE Cal÷/DPG = 0.13 (mot/moll Egg PG/soya PE/Ca 2+ 70-85 mol% soya PE Ca 2+/egg PG = 0,25-1 (raol/mol) Soya PS/soya PE/Ca 2. 70 mol~ soya PE Ca2 +/soya PS = 0.25 (real/real) Soya P I / ~ y a PE 85-90 motq[ soya PE Dipalmit oleoyl-PC/st a micidin gramicidin/PC = O.l (mol/mol) Total lipids from rod outer segments Total lipids from inner mltochonddal membranes Total ph~pholipids from Escherwhia cob membranes

Water ~llten|

Temper- Ref.

(wt~l

IttuI¢ (°C)"

96

60

90

96

60

90

80

45

107

94-97

55

103

85-90

30

i86

86-93

O

110

10

lll

80

45

107

85

20

187

95

30

113

95

30

188

30

lgg

96

30

19¢/

96

25

118

95

4

119

4

191

25

122

• Low~! temperature for the formation of.the isotropie phase. b [~lal~l from A. /aidtawJi.

Isolated from bovine heart.

247 water contents, A cubic phase is probably formed also ai ve.~ high water contents if the acyl chains have six cis double bonds (Table ll), since a transition from an Lo to an isotropic phase is observed between 60 and 90°C; above 9 0 ° C only a narrow symmetrical N M R signal is obtained. Considering the shape of this PC molecule it is not surprising that a cubic phase may form. Moreover, if PC is mixed with equimolar amounts of cholesterol a cubic phase is most likely obtained with PC species containing acyl chains with two, three, and four e/~ double bonds as welt; cholesterol is known to promote the formation of nonlamallar phases in several membrane llpid/water systems [70,106]. A freeze-fracture electron micrograph of a dilinoleoyI-PC (DLiPC)/cholesterol sample (heated to 80 °C) showing three-dimensional arrays of LIPs, in addition to vesicles [90], supports the proposal that the above-mentioned systems form a cubic phase [30]. The phase diagram of the D O P C / M O / w a t e r system exhibits a large phase area representing a bicontinuous cubic phase [16] (Fig. 3). A cubic phase seems to be obtained [107] with DPlPC/MG and D P P E / M O as well (Table II). These ob~ervatinns are of int~'rest since MO is known to be a very effective fusogenic lipid [108] and they glv¢ further support to the recent suggestions that isotropic lipid structures are a prerequisite for memheane fusion to occ~:r (subsections V-C and VIII.A). Cubic phases are often formed when one lipid formlng an L a phase, and one lipid forming an Hal phase, are mixed ir~ certain proportions together with water [109] (Fig. 7; Table I). Several such mixtures are 5sted in Table II and it is therefore likely that they form cubic phases. Thur, narrow 31P-NMR signals can ha obtained when egg PC or DOPC is mixed with large amounts of MGhiDG, soya PE or DOPE. An eqinmolar D O P C / DOPE mixture gives rise to narrow N M R signals both in the absence and the presence of cholesterol, although cholesterol considerably lowers the temperature for the appearance of this signal [110], The presence of a cubic phase in the different P C / P E systems is strongly supported by the results presemed in Table I concerning P C / P E mixtures. The system DLiPE/dielaidoyI-PE (DEPE) is another example where a cubic phase may occur (Table II). DLiPE forms and H u phase above - 1 5 0 C while DEPE forms an L . phase up to 60*C [111]; mixtures of these lipids form a phase structure allowing isotropic motional averaging over a broad coneantration interval. Finally, a DOPE/doliehol mixture containing 25 mol% dolichol seems to form a cubic phase since a three-dimensional array of LIPs is seen in freeze.fracture electron micrographs from this mixture [1121. In Section V1 it was concluded that only two exampieS are known of anionic membrane lipids forming a cubic phase. One example is DPG which is able to form a cubic phase together with the cadonJc local anaesthetic

dibucaine (Table i). De Kruijff et aL [72] have suggested that a cubic phase is formed by the D P G / C a -'÷ system. A ~IP-NMR spectrum of this system exhibits only a narrow symmetrical signal, and an interwoven network of lipid and water fracture planes wa~ obtained in freeze-fracture electron micrographs. The lipid-fracture planes appear to be interconnected with sizes of about 100-200 nm, and no defined diffraction bands could be observed with X-ray |187]. These data are, however, also compatible with another structural interpretation, namely the so-called L~ phase which can be considered as a "melted cubic phase" (subsection IV*A). It is probable that the anionic membrane lipids in several cases can participate in the formation of stable or metastable isotropic structure~, when mixed with a lipid forming an H u phase. A narrow symmetrical 3tP-NMR signal, together with spectral components arising from L. and H u phases, can be obtained from mixtures of PE with DPG, phosphafidylglycerol (PGL phosphatidylserine (PS) or phosphatidylinositol (Pl); in most cases a low ratio of CaZ+/anionic lipid is needed to obtain this N M R signal (Table !I). The fraction of the anionic lipid in these mixtureS is between 10 and 30 tool% which is approximately the fraction of ionic lipids occurring in a biological membrane. The P E / D P G system is of special biological interest since these 5pids probably are the major components of the inner monniayar of the inner mitoebendrial membrane 1113l. It is noteworthy that a dioleoylphorphatidic acid/chlorproma?Jne mixture (molar ratio l : l ) probably forms a cubic phase since freeze-fracture electron micrographs of this mixture show a three-dimensional array of LIPs

I1"141. The influence of membrane proteins and hydrophobic or amphiphili¢ peptides on the phase equilibria in membr~_,t¢ lipid/water systems is of great interest. It has been shown that eytoehrome c, a peripheral protein associated with the inner mitochondrial membrane. forms isotropic and H u phase structureS together with a total lipid extract from mitochonchia [115], with DPG. and with mixtures of DPG and PE or PC [113]. Glycophorin, a major integral protein from the human erythrocyte membrane, has a bilayer-stabiliziog effect on DOPE [116], and gramlcidm, a hydrophobie peptlde often used as a model for ;,~tw.ral membrane proteins, induces the formation of an H n phase in DEPE/water, POPe/water, DOPC/water and DLiPC/wates systems [48,117]. Gramicidin has been suggested to form a cubic phase together with dipalmitoleoyl-PC (Table 11). A freeze-fi-acture electron micrograph obtained from this system [llSJ resembles the mlcrograph of D P G / C a z* (see above). Therefore, it is possible that also the gramicidin system forms an L a phase ('melted cubic phase'). The polar lnembrane lipids from the bacteria A. laidlawii and & solfararicus can form a cubic phase

248 (Table [). Narrow symmetrical 31P-NMR signals have also been recorded from some total membrane lipid mixtures (Table II). Rod outer segment lipids give rise to a narrow signal already at 4 ° C according to De Grip et al. [1191. However, Deese et al. [120] found that these lipids form just an L~ phase up to at least 45°C. Albert ct al. [121] seem to have explained the conflicting resuits; the rod outer segment lipids form isotropic structured and H n phases in the presence of more than 4 mM Ca 2÷ (De Grip ct al. [119] used 2 mM Ca 2+ and 3 mM Mg2+), while just an L , phase is obtained in the absence of Ca 2+ (the conditions used by Deese et al. [120]). A substantial fraction of the total lipids from the inner mitochondrial membrane forms a structure allowing isotropie motional averaging at 4 ° C (Table II); this fraction is further increased at 37°C. The total phospholipids isolated from Escherichia coil membranes form a lamellar phase at 4 5 ° C in the absence of NaCI, but addition of 100 mM NaCI (more close to the situation found in the cell) makes almost all the lipids form a structure allowing isotrepic motional averaging at 37 ° C (Table 11) [122]. A cubic phase is probably formed at 7 0 ° C in the presence of 5 M NaCI by the total polar membrane lipids isolated from the extreme hahiphile lfalobacleriurn cutirubrum, as indicated by the appearance of three-dimensional arrays of LIPs on free, efracture electron mierographs [123]. Finally, narrow symmetrical 31P-NMR signals have b~en obtained f~om some intact biological membranes. The phosphofipids in microsomes isolated from rabbit, beef and rat liver are mainly in a bilayer arrangement at 4 ° C , but a large fractkn of the lipids experiences isotropic motion on the N M R time scale at 3 7 ° C [124-126]. In contrast, an aqueous dispersion of the total lipids extracted from rat liver mi¢rosomes form a bilayer structure. A rather narrow 31P-NMR signal was obtained even from mierosomes prepared from the smooth endoplasmic rcticnium which excludes the possibility that the narrow N M R signal is completely derived from RNA phosphorus [124]. The phosphofipids in the cytoplasmic membrane of E. coil form a bilayer structure at .25 ° C but a fraction of the lipids undergoes isotropic motion on the N MR time scale at 37 ° C [122]. It is, howcvcr, less probable that the lipids form a macroscopic cubic phase in these cases; instead local rearrangements of the lipids to structures allowing isotropic motinnal averaging are more probable. VIII. Biological relevance of cubic phases and isotropic structlwes VIII-A. Membrane fusion Membrane fusion is a very important phenomenon in all cells. In particular it is involved in processes of transport in which membranes encapsulate different cell

substances often called membrane traffic. Other examples are in sperm-egg fusion, where life starts and virus-cell fusion, which leads to infection. Several recent reviews on membrane fusion are available [58,68.127129]. The molecular mechanisms behind the fusion process are poorly understood, and a number of various factors arc discussed in the reviews cited above. Here we will confine ourselves to the fusion occurring in lipid systems, in particular systems where isotropie Stl~dctures probably are involved in the process [58,68,129-13!]. It is quite clear that in the course of fusion structural reorganizations have to occur in the lipid bilayers, so that for example two lipid vesicles can form one new larger vesicle. Thus, the essential step in fusion is the rearrangement of the lipid molecules of the two apposed membranes to form a single, continuous membrane. In order to investigate fusion both the methods used and the lipid systems chosen are very critical as has been discussed by Elleas et at. [130,131]. For an understanding of the fusion process, of course, both dynamical (kinetics) and structural aspects have to be considered, but here only the latter one will be briefly discussed. Thus, the lipid phase structure, and correlation of nonleaky fusion with phase transitions of macroscopical systems, plays an important role in this respect. Using vesicle made of DOPE-Me Ellans ut at. [130] found increased fusion (Fig. 23) to occur in the temperature range in which isotropie structures were formed in dispersions of this lipid [132]. It is very likely that this isotropic structure corresponds to a cubic liquid crystalline phase, as indicated by the recent X-ray diffractiop studies by Gruner ct at. [18] (Table I). When the temperature was above the transition temperature for formation of the H n phase, lysis of the vesicles developed. A prareqalsite for the membrane fusion to take place is the formation of intermembrane structures. These structures are most probably similar to the LIPs observed by EM [68] and the structural units of cubic phases observed by X-ray diffraction and N M R (Section V). The theoretical works by Siegel [55-58] strongly indicate that a sequence of intermcmbrane structure,s are involved in the mechanism of the L J H u phase transitions and these account for most of the NMR, DSC and EM data concerning these transitions. The postulated intermediates are consistent with the models for the relative chemical potcetials of reversed-phase structures [49,133[ and also with X-ray diffraction on these systems [18,52,65,66]. From theoretical calculations Siegel [55-57] suggested that several fipid systems exhibiting L J H n phase transitions should form a type of intermediate that would produce isotropic structures in a temperature interval starting tens of degrees below the L J H n phase transition temperature (TH). Examples of such systems are DOPE-Me or D O P E / I X ) P C tmxtures [18], and DPPC/alkane or POPC/alkane dispersions

249

i '° <

,0

~0

uoPt.,

<_

,.4s

20

40

:

i

60

8e

TEMPERATURE ( ° C ) Fig- 23. I n i t i a l r a t ~ o f H *-induced fusion a n d leakage o f D O P E - M e

lip~mnumas a f~tinn of temperature.The inset shows the temperature dependence of the initial rates of fusion and leakage of DOPE/DOPC 12:1) llposomes. Tt and TH ~re the temperature intervals whele the cubic and HII p h ~ dominale, rcspectivd3 From ReL 130.

[17I. The model of the L . / H n phase transition mecbeproposed by Siegel is shown in Fig. 16. Fhictuations, beginning at temperatures well below the T H, lead to the formation of intermembrane micellar intermediates such as IMIs, so that the lipids now can exchange in the outer monolayers. According to Siegel the initial step in a phase transition is the formation cf these very short-lived reversed micellar structures. These miceUes may than aggregate in the plane of the two apposed membranes and transform either t o a n H . phase or to a new intermediate called interlamelhir attachment (ILA), which is very long-lived compared with the reversed micelles. The ILA is suggested to have a structure ~ a l l a r to die unit cell in some bicontinuous reversed cubic phases [eL Figs. 9 and 16). The conditions for the formation of ILAs are given a very simple form in Siegels' theory (see also subsection IV-B). The probability for an ILA to form largely depends upon the experimental parameter Z = a j a r , , where a L is the average polar head group area in the L . phase just below Tu, and a n is the average polar head group area per molecule in the H . phase just above T u. The

parameter Z is closely related to the spontaneous radius ef curvature (Ral in Gruners" theory [49[ and to the packing parameter introduced by Israelaehvili et al. [461. Now. if Z is less than 1.2 or analogously, with 'intermediate" values on R o the IMIs can form ILAs. which in turn should res);It in membrane fusion with non-leaky mixing of the aqueous contents (Fig. 16). This model does not predicz the formation of ILAs for every lipt~ exluhiting an L ~ / H u phase transition. Narrow peaks in the N M R spectra are not always found for lipids which can form the H u phase 117.951. However. Shyamsunder et al. [59] showed that the DOPE/water system (between about 30-60 wt% water) can form a reversed cubic phase (Table I) by cycling the temperature above and below T u several hundred times. It has been observed in other PE/water systems that LIPs can be seen on EM micrcv~aphs during the transition between LQ and H n phases, although no arrow symmetrical peaks are observed in the JLP-NMR spectra [68,134,135]. Siegel's theory [55-57] shows that it is difficult for the ILA to revert to the IMI structure. although the IM1 can easily go back to apposed bilayers. This explains the gradual formation of the cubic phase structure [59], since in each cycle through T x the accumulation of more ILAs occurs. Maybe this technique can be used to form cubic phases in any lipid/ water system where H u phases can form. However, a correlation between the fusion event and the isotropic state exists only when isotropic lipid structures are fok.'med on the li'st heating scan [1311. Recently, Siegel et al. [1~61 showed that small amounts of" diacylglyeerols (2 mol~) added to a phosphohpid system lead to vesicle fusion with non-leaky mixing of aqueous contents. The mechanisms behind this fusion was correlated with the formation of ILAs responsible for the i~.ntropic motion on the N M R time scale, It was also reported ~hat 2 reel% of diacylglycerols, which are lipid species produced by the PI-eycle in vice [93], can decrease the temperature by about 2 0 ° C for the onset of the formation el isotropic structures and the rapid membrane fusion ili the DOPE-Me system.

VIII-B. Regulation of lipid composition in biological membranes Cubic and isotropic phases can be formed by single lipids, binary lipid mixtures, and total lipid mixtures isolated from biological membranes (Tables I and It). Lipid structures allowing isotropic motional averaging are formed even in intact membranes, although, with the exception of microsomes, to a much lesser extent than with the extracted lipids. It can therefore be assumed that membrane lipids, at least in metabolically active membranes, must have the possibility to rearrange to isotropie structures in order to fulfil certain

250 functions. However, nonbilayer structures are not allowed to occur permanently in a biological membrane since this would destroy its barrier properties. Instead, the membrane !ipids should be in the "vicinity" of a phase transition under physiological conditions so that Ioc:ai and transient nonbilayer structures can form easily in response to some triggering mechanism. Consequently, it is reasonable to assume that the balance between bilayer and nonbilayer lipid structures is actively regulated in cells growing under different environmental conditions. Experimental results obtained from four bacterial species [11,137-141] show that this assumption seems to be true. The regulation of membrane ~ipid composition and the physicochemical properties of the membrane lipids have been intensively studied in A. laidlawii. The membrane of strain A of this organism contains six amphiphilic lipids, three 1-.oninnie and three ionic 147[. MGhiDG and DGhiDG are the major lipids and they account for between 55 and 75 mol%. The polar head group composition of the lipids is affected by several factors: the growth temperature; incorporation of different fatty acids into the lipids; and incorporation of sterols, hydrocarbons, alcohols, detergents, and chlorophyll into the membrane [47,137,142-144]. All these factors affect the molar catio M G I u D G / D G h i D G . MGIuDG torms cubic and It a pha~es above 1 0 ° C while DGIuDG and the other lipids form a lamellar phase [11,145]. The following eonehision was drawn: when the growth conditions of the calls are changed in such a way that the phase eqnifibria of the membrane lipids are shifted towards nonbilayer structures, the cells respond by changing the ratio M G I u D G / D G I n D G in order to shift the phase equilibria back to the bilayer structure [47]. The formation of the nonlamellar phases by A. laidlawii lipids depends critically upon the concentration of MGIuDG [1-ll. The regulation of the 'bilayer stability" is exemplified in Fig. 24, which shows the phase equilibria of total lipid mixt::res isolated from A. laidlawii. The lipids contain different proFortions of paimitoyl and oleoyl acyl chains, When the proportion of the oleoyl acyl chains is increased, without changing the polar head group composition, the phase equilibria are shifted towards the cubic and H H phases [146,149]. Howvwer, by lowering the molar ratio M G I u D G / D G h i D G the transition to the nonlamellar phases begins at approximately the same temperature ( 1 0 - 1 5 °C above the growth temperature) in all the lipid extracts. A cubic phase appears in all the lipid mixtures, with the exception of the mixture with the lowest proportion of oleoyl acyl chains, which transforms directly from an L~ phase to an Hti phase. Aceor,lingly, two kinds of restriction seem to determine the membrane lipid composition in the A. Imdlawii cells: firstly, a large proportion of the lipids are not allowed to enter the gel phase since this

MGDO/OGOG,at,o

,*o

20

40

Auyl c r a m compusolon

60

80

100

4~1BI1¢ r e e l / m o l l

Fig. 24. Phase equilibria of total lipid mixtures, COnlaininlidifferent amounts o" ?almltoyl and oleoyl chains (l~er x-axis), from membrane~ of A. Ioidlawil grown at 37°C. Water ~ntents were appr~. 20 wt~, The upper x-axis shows the metabolically obtained MGluDG/DGIuDG ratio. The lower hatched area denotes the gel to liquid crystallinephase transition interval as determined by electron spin resonance. The upper hatched area denotes the appearance of nonlamellartH a and/or cubic) phases in the lipid mixtures. Adapted from Ret. 11 will inactivate the membrane-bound enzyme and transport activities [150]; and secondly, nonbilayer fipid phases are not ~llowed to form permanently, since this will break down the barrier properties of the membrane. Preliminary 2H. and 31P-NMR studies on intact A. laidlawii membranes show that a narrow symmetrical signal originating from the membrane lipids appears in spectra recorded at 50oC (Rilfors, L., Lindblom, G., Wieslander, ~. and Bremiel, I., unpublished data). Similar experiments to those described above have recently been performed with Closlridium butyricum [138,141]. The major membrane fipids in this organism are PtL plasmaenytethanohimine (PIaE), and the glycerol acetal of PlaE (GAPIaE). T h e ratio (PE + PlaE)/GAPIaE is decreased when the degree of aeyl chain unsaturatioo in increased. The PE plus PlaE fraction forms an H , phase above 0 ° C while GAPlaE forms an L a phase up to at least 5 0 ° C . A P E / P l a E / GAPIaE mixture (33 wt% GAPlaE) forms a fipid stracture at]owing isotropic motional averaging above 30 ° C. Similar regulatory mechanisms thus seem to operate in A. laidlawii and C. bmyricum although the membrane lipids involved differ completely. The membrane lipids in B, megaferium are PE, PG and DPG, and the fipids contain mainly iso and anteiso methyl-branched, saturated aeyl chains [151]. When the growth temperature is increased the fraction of PE remains fairly constant (55--65 molCg), while the molar ratio iso/anteiso aeyl chains is markedly increased. PE isolated from B. megaterium grown at 20 ° C have a low ratio of iso/anteiso acyl chains and begins to form a cubic phase at 50 ° C at low water contents (Table 1). PE isolated from cells grown at 550C have a lO-fold higher

251 value of the ratio iso/anteiso ac~,l chains and form an L~ phase up to at less 6 5 ° C [139| (Fig. 2i). Since an increase in the temperature will increase the tendency of PE to form cubic and H n phases [106], the temperarere-induced regulation of the acyl chain composition in R megaterium membranes is most probably necessary in order to maintain a stable lipid bilayer. The phase equilibria of PE isolated from Pseudo,. monas fltwresce~., grown at 5 and 22°C have also oeen investigated [140]. PE constitutes about 75% of the phospholipids in the membrane of this organism, the remainder being mainly PG and DPG. The proportion of unsaturated acyl chains in PE was decreased from 76 to 65~ when the growth temperature was raised from 5 to 22°C. PE (5°C) formed a mixture of L= and H u phases from the growth temperature up to 43°C; above this temperature just an H u phase was present. PE (22°C) formed an H n phase at 65°C while a mixedphase system was formed between the growth temperature and 65°C. Surface pressure-area curves for monolayers of the two PE preparations remained unchanged down to 5 ° C which indicates th~tt the gel to liquid crystalline phase transition temperature is below 5 ° C for both the preparations [140]. The aheration of the acyl chain composition therefore seems not to he needed in order to regulate this phase transition temperature. Instead, the increased tendency of PE to form nonlamenar phases at higher temperatures is counteracted by regulating the acyl chain composition, and the balance between the lamellar and nonlamellar phases is thus kept approximately constant [140]. Many organisms, both prokaryraic and eukaryotic, regulate their membrane lipid composition in response w various changes in the environmental conditions, such as the ambient temperature, supply of fatty acids, ionic conditions, and the presence of sterols, hydrocarbons, and alcohols in the membrane [106]. Nearly all biniosieal membranes that have ~ean investigated contaln at least one 'Apid species [152,153] that can form a cubic a n d / o r an Hll phase, and the formation of these phases is dependent on all the factors mentioned above. It is therefore possible that one aim with the lipid regulation mechanisms is to keep the balance between the bilayer and nonbilayer lipid structnses on an appropriate level. It was shown in subsection IV-A that the packing parameter or the spontaneous curvature plays a dominant role for the structure of the lipid aggregates formed. Front our studies on A. laidlawii we have suggested [11.47} that the lipid composition in a biological membrane is regulated so that an optimal packing is maintained in the membrane. Recently, Gruner et al. [tg] proposed that the membrane is striving towards a constant spontaneous radius of curvature of the lipid mmmlayer, an idea also partly based on our experimental findings on A. laidlawii membranes. This

latter view has advantages, sillc~ ~h:s model contains quantities that probably are more directly measurable. However. the drawback with this phenomenological approach is that the molecular elemen.t is less pronounced. Furthermore. there are some experimental indications that an L 3 phase (subsection IV-A) may form with membrane lipids. Thus, at high water contents the typical isotropic phase behaviour of an L~ phase has been observed for samples containing the total lipids extracted from A. laidlawii membranes [11[, again suggesting that the bilayer curvature is an important factor for the structure of a biological membrane. The generality of the above-mentioned hypotheses is currently investigated. V I I I - C Membrane structures

Since cubic and isotropic phases ave formed by total lipid mixtures extracted from biological membranes, and since lipid structures allowing isotropic motlonal averaging seem to occur in intact membranes (Tables I and II), it is conceivable that membranes, and organelles consisting of membrane networks, form structures resembling cubic phases. Below we will discuss three such example~ Etioplasts are found in leaves of plants grown in the dark and after the exposure to fight they transform into chloroplasts. The etioplast contains a ~ghly ordered, branched tubular membrane structure (Fig. 25) called the prolamellar body (PLB) which develops into the thylakoid membranes of the chloroplast in the presence of light. The thvee-dinlelnsional arrangement of the tubules in the PLB has been investigated by several authors

Fig, 25. Ptolamellar body in elloplasts isclated from wheat lu~es, The b l = k bin- r~prlr~l:lllS ~ 0 nm. By comlesy of Dr. E. Selslam.

252 (Ref. 15,~ and references therein). The basic unit of most PLBs is a tetrapodal structure which has four short tul:'tlar arms (30-35 nm in diaraeler) meeting at one point with telrahedral angles. This is similar to the arrangement of the structural elements in a hieontinuous cubic phase belonging to the space group Pn3 or Pn3m [27]. A fact favouring the possibility that the PLB has a bicontinuoas cubic structure is its lipid composition: the gaiaelolipids MGalDG and D G a l D G account for approx. 75-80 mol% and the ratio M G a l D G / DGalDG is high (1.6-1.8) [155-157]. Galaetolipid mix. tares with this ratio can form a cubic phase (Fig. 7). Moreover, one pigment-protein complex dominates the protein content of the PLB, the NADPH-protochlorophyllide oxidoreducease (EC 1.6.99.l) [154,157,158]. The accumulation of this pigment-protein complex is unique for the PLB and may facilitate or induce the formation of this structure [156]. Israelachvili and Wolfe [159] have shown that the branched PLB membrane structure and the planar thylakoid membranes have similar average curvature and inside.outside surface areas. The membrane surfaces of the PLB may therefore represent one of the constant-mean-curvature surfaces occurring in some bieontinuons cubic phases (subsection Ill-C). This iwplies that the packing and molecular organization of the iipids and proteins can be similar hi the PLB and thy]akoid membranes, and that the translormation between them need not to involve energy requiring 'flip-flop' of membrane components from one side to the other. The second intracellular membrane network discussed will be the endoplasmic relieuhim (ER). Two functionally distinct regions of E R exist: (1) the rough ER which in most cell types forms wide flattened sacs ananged in a compact parallel array; however, in some cell types the rough ER forms a three.dimensional network of tubular elements: and (2) the smooth ER which as a rule forms a three-dimensional network of fine tubules with a diameter of 30-60 nm; the tubules are thus of about the same size as those building up the PLB [160,161]. It may be speculated that the networks of tubules resemble a bicontinuuns cubic phase, in which proteins are embedded (Anderson, D. el al., unpublished data). Support for this speculation is given by the narrow symmetrical signals that is seen in 31p. N M R spectra obtained from liver microscopes [124-1261. Finally, a tentative model for the structure of the plasma membrane of the bacterium S. solfataricus has been presented [162]. The total membrane lipids from this organism form a cubic phase belonging to the space group Pn3m under physiological conditions (Table I). The plasma membrane structure proposed by Luzzati and co-workers is built up by hpid bilayers arranged in such a way that two mutually intertwined and unconnected three-dimensional water networks are formed:

one of the networks communicates on one side with the cytoplasm and is plugged on the other si'Je by proteins from the envelope surrounding the cell, whereas the other network is open to the extraeellu]ar medium through the pores of the protein envelope and is closed by lipids or proteins on the cytoplasmic side (Fig. 8 in Ref. 162). Luzzati and co-workers also suggest that the metastability observed for the cubic phase could protect the organism against the thermal fluctuations to which it can be exposed. If the temperature is decreased the high-temperature structure is metastably preserved [08,162]. V l l l - l ~ F a t digestion In most vertebrates the fat digestion occurs rapidly in the upper small intestine through the integrated action of pancreatic lipase, colipase and bile. Cofipase binds to the surface of fat droplets in the presence of bile salts and provides an attachment site for fipase (see Ref. 163 for a review). Lipase attacks two of the ester bonds of the triecylglycerol molecules, producing first a diacylglycernl and one fatty acid and then a monoacylglycerol and another fatty acid. Neither the substrate nor the produces are soluble in the reaction medium and the only substance able to form a fiquid crystalline phase by itself is the monoacylglyceroL During the fat digestion a number of different lipid phases are formed, among other lamellar and cubic liquid crystalline phases [86]. These studies were performed under simulated physiological conditions and it was found that the cubic phase was composed of approx. 45~0 each of monoacylglycerols and protonated fatty acids, the rest being diacylglycerols and tri. acylglycerols. Olaic acid and M O form a viscous isotropic phase of unknown structure when mixed in equal proportions in the presence of excess water, and the cubic phase of monoacylglycerol/water can solubilize about 5~o triacylglycurol [164]. It seems resonable to speculate here that Nature betraying good insight prefers the cubic phase in front of a ndcellar solution or any other fiquid crystalline phase, in order to effectively carry out the work of fat digestion. Lipeses perform their enzymatic reaction with highest efficiency at the water/hydrucarbon interface of a lipid bilayer [165,166]. A hicontinuous cubic phase formed by lipid bilayers might therefore constitute a suitable matrix for the enzymatic process: (1) the substratus can be incorporated into the cubic phase and will thus have a proper location for the lipulytic attack, (2) large interracial surfaces for the fipases are created in a small volume by the cubic phase, and (3) the products are effectively "stored' in the cubic phase. A cubic phase of monoacylglycerul/water can be dispersed by sodium chohite, but it is not known whether die

253

cubic phase is directly involved in the mucosal uptake of lipids [164]. VIII-E. A ctivity of membrane-bound enzymes

Membrane-bound enzymes must in general be surrounded by membrane lipids in order to be active [167]. Several investigations show that the efficiency of protein incorporation dining the reconstitution process 6125,168-170] and the activity of membrane-bound proteins and enzymes [170-177] is enhanced in the presence of fipids forming nonbilayer structures, or by the incorporation of molecules into the membrane known to destabilize the bilayer structure. The results in some of these investigations indicate that an optimal protein incorporation, or an optimal enzyme activity, is obtained with lipid mixtures able to form isotropic lipid phases. When a Triton X-100 extract of human erythrocyte membrane proteins was reconstituted with mixtures of egg PC and soybean PE the protein/lipid ratio of the reconstituted vesicles was maximal when egg PC accounted for between 15 and 35 wt% of the lipids [168]. These lipid compositions give rise to 3tP-NMR spectra with a narrow symmetsieal component and freeze-fracture electron micrographs exhibiting LIPs [178]. The activity of Ca2+-ATPase isolated from sarcoplasmic reliculum was studied when reconstituted in mixtures of egg PC, soybean PE and diolein or dipalmitin [175]. The Ca 2+ uptake activity and the coupling ratio (Ca 2+ uptake activity/ATPase activity) were much higher in mixtures containing diolein; a large narrow symmetrical component was seen in 31p-NMR spectra recorded from these lipid mixtures. Jensen and Sehutsbach concluded that the phase properties of the lipids are mere important than the polar head group specificity for the activity of reconstituted dolichyl-phosphomannose symhase [173,174]. Although MGaiDG, which forms an H u phase under physiological conditions [9], alone supported enzyme activity, higher activities were observed in M G a l D G / D G a l D G mixtures containing between 2.0-40~ DGalDG. Such lipid mixtures can form a bicontinuous cubic phase (Table I; Fig. 7) and give rise to three-dimensional arrays of LIPs in freeze-fracture electron mierographs 171,971.

maticn can be obtained on the aggregate structure; EM visualizes intermediate structures at the phase transitions, and TEM directly gives the dimensions of the phase structure. Cubic pha s ~ formed by biological membrane lipids are in general bicontinuous (some lysolipids are exceptions). Their structure and formation are dominated by curvature energies and therefore constant-mean-curvature surfaces and differential geometry have been shown to offer valuable tools for their description. It is well established that several biological membrane lipids can form cubic liquid crystalline or isotropic phases. From a biological viewl~int it is of special interest that mixtures of phosphatidyicholine and phosphatidylethaoolamiue, of monog~actosyldiacylglycerol and digaiactosyldiacylgiycerol, and of phosphatidylethanolaminv and different anionic fipids are able to form cubic phases and isotropic structures at high water contents, since these lipid pairs occur very frequently in the membranes of both eukaryotes and piokaryotes. Very few suggestions have been presented where a reversed hexagonal phase, another phase structure formed by many membrane lipids and lipid mixtures, has a biological fuuedon. Tight junctions between epithelial cells have been proposed to consist of lipid cylinders of the type building up a reversed hexagonal phase [179]; however, this suggestion was later rebutted {102,193]. On the other hard, several argumuets make it probable that cubic and isotropic lipid structures am the nonbilayer structures that are of greatest biological relevance: (1) low amounts of energy are required for the small structural reorganizations of lipids and protsins that occur during the transformations between a lamenar phase and a bicontlnuous cubic phase represented by a minimal surface or a constant-mean-curvatore surface; (2) the nonbilayer phases and structures formed by the total lipids extracted from biological membranes, and by intact membranes, are predominantly cubic phases or isotropie fipid structures; and (3) existing experimental data concerning membrane fusion, regulation of membrane lipid composition, the structure of the prolameflar body, and fat digestion and absorption point more or less directly to an involvement of cubic a n d / o r other isotropic structures.

A

~

IX. Com:laslans Various methods must be utilized in the study of cubic liquid crystalline phases and isotropie structures, in particular X-ray diffraction, N M R and EM. Briefly, the strength with these techniques is: X-ray diffraction allows a determination of the space group and the dimensions of the phase structure; N M R can dislingnish between a bicontinuous phase structure and a structure built up of dosed lipid aggregates, and infor-

We are grateful to Dave Anderson, Sol Gnmer, Afie Verkleij and Hkkan WenneretriSm for stimulating discussions. Dave Anderson, Eva Selstam, Ishi Talmon and Arie Verkleij are also thanked for supplying the pictures in Plate I and Fig. 13, Fig. 25, Fig. 17, and Fig. 15e. respectively. We acknowledge Dave Anderson, Joe Bemz, Sol Gruuer, Dave Siegel and Ishi Taimon for sending us unpubfisbed results. This work was supported by the Swedish Natural Science Research Court-

254 oil Finally we appreclatc the excellent treatment given us b y the Ladies at IG'onlund, H~illn~is, where a large part of this review was created. References 1 L u ~ t i , V. (1968) ip Biological M ~ b ~ n ~

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