Hydration Properties of Lamellar and Non-Lamellar Phases of Phosphatidylcholine and Phosphatidylethanolamine

CPL CHEMISTRY AND Chemistry and Physics of Lipids PHYSICS OF LIPIDS ELSEVIER 81 (1996) 117 131

Hydration properties of lamellar and non-lamellar phases of phosphatidylcholine and phosphatidylethanolamine

Thomas J. McIntosh*

Department ~l Cell Biology, Duke Unicersity Medical Center, Durham, North Carolina 27710, USA

Abstract

Two of the most common phospholipids in biological membranes are phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Over a wide range of temperatures the PCs found in biological membranes form lamellar (bilayer) phases when dispersed in excess water, whereas PEs form either lamellar or hexagonal phases depending on their hydrocarbon chain composition. This paper details the hydration properties of lamellar and hexagonal phases formed by PCs and PEs, focusing on the energetics of hydration of these phases. For the hexagonal phase, the energy of bending the lipid monolayer is a critical term, with other contributions arising from the energies of hydrating the lipid headgroups and filling voids in the interstices in the hydrocarbon region. For the lamellar phase of PC, the water content is determined by a balance between the attractive van der Waals pressure and repulsive hydration and entropic (steric) pressures. In the case of PE bilayers, recent experiments demonstrate the presence of an additional strong, short-range attractive interaction, possibly due to hydrogen-bonded water interactions between N ' H3 groups in one bilayer and the PO4 groups in the apposing bilayer. This additional attractive pressure causes apposing PE bilayers to adhere strongly and to imbibe considerably less water than PC bilayers.

Keywords: Lamellar and hexagonal phases: Hydration pressure: Steric pressure; Hydrogen bonding: X-Ray diffraction

1. Introduction temperature ranges when dispersed in an aqueous phase [1,2]. However, other phospholipids can Biological membranes contain several classes of form non-bilayer (non-lamellar) phases in excess phospholipids. Many of these lipids, such as the water. In particular, the second most common most common membrane phospholipid, phos- membrane phospholipid, phosphatidylethanol- phatidylcholine (PC), form bilayers over wide amine (PE), can form either lamellar or hexagonal It phases depending on a number of factors, including temperature, water content, and the *Tel.: +1 919 684 8950: fax: + 1 919 684 3687. composition of the PE hydrocarbon chains [3--7].

The presence in membranes of lipids that tend Tartar [14] considered micelle tbrmation by to form non-lamellar phases has long intrigued paraffin salts and found that the shape of the many membrane biophysicists. Numerous theo- micelle depended on the fully extended length of retical and experimental investigations have con- the hydrocarbon chain and the area occupied per sidered the role of these lipids in membrane polar headgroup. Tanford [15,16] considered am- structure and dynamics, as well as a variety of phiphiles with two chains, such as phospholipids, membrane activities such as enzymatic reactions and pointed out that the value of area per and membrane fusion. Many of these studies are molecule is lower ['or bilayers than lbr micelles reviewed in other articles in this special issue. This and that bilayers can accommodate a limitless review focuses on how diacyl phospholipids which number of chains without requiring a change in form non-lamellar phases impact the fundamental molecular area. Israelachvili and colleagues interactions between surfaces in water. Specifically [17,18] stressed the importance of molecular shape we compare and contrast the hydration properties on lipid organization. They argued that bilayers and intersurface interactions of lamellar and are formed by cylindrically shaped lipid molecules hexagonal phases containing the two most thor- (excluded area in the headgroup region approxi- oughly studied phospholipids, PC and PE. The mately the same as in the chain region), whereas factors determining the energetics of dehydrating non-lamellar phases are formed by cone-shaped bilayer and hexagonal phases are discussed. Em- molecules. Specifically, lipid molecules with the phasis is given to recent experiments that have headgroup area smaller than the hydrocarbon revealed the mechanisms by which PE affects the chain area would be expected to form inverted hydration of membrane bilayers. hexagonal phases. This shape hypothesis is supported by the observation that highly unsaturated 2. Phase properties of PE and PC PEs and long-chained PEs (which would be expected to have large volumes in their hydrocarbon Membrane phospholipids contain a mixture of regions) form hexagonal phases, whereas short- fatty acid chains, varying in length and number of chained PEs form lamellar phases [3,4], Further double bonds. In general, diacyl PCs found in experimental support for this hypothesis has been biological membranes form lamellar phases in obtained with several electrically neutral lipid spe- excess water at physiological temperatures [1,2]. cies with systematically varying headgroup vol- Diacyl PEs with short (12 carbons per chain) umes [19]. In addition, the incorporation of lipids saturated chains also tend to form lamellar phases with polyethylene glycol (PEG) covalently at- at physiological temperatures [4,8], whereas PEs tached to their headgroups stabilizes the bilayer with unsaturated chains tend to form hexagonal phase in DOPE/cholesterol mixtures, at least II phases [3-7,9,10]. For fully hydrated lipids, the partly because of the complementary 'inverted transition temperature for the lamellar-to-hexago- cone' shape of the PEG-lipids [20]. nal phase transition depends on the hydrocarbon Gruner and colleagues [21 23] noted that the chain length and number of double bonds per concept of molecular shape ignores other factors chain. For example, fully hydrated dioleoylphos- such as headgroup charge and intermolecular hy- phatidylethanolamine (DOPE), which has hydro- drogen-bonding. This is an important consider- carbon chains containing 18 carbons with one ation in terms of the PEs, because it has been double bond, has a lamellar-to-hexagonal phase shown that at high pH, where the PE headgroup transition near 5°C [11,12]. is negatively charged, the hexagonal phase is con- The factors that determine the phase behavior verted to single-walled vesicles [24]. Moreover, of particular lipids have been investigated for intermolecular hydrogen bonding is thought to many years (see review by Seddon [13]). Here we play a role in the structure and properties of PE consider some of the basic principles that are phases [7,10,25 31]. Gruner and colleagues important to the hydration properties of hexago- [22,23] view the transitions between mesomorphic nal and lamellar phases. phases with curved interfaces in terms of a "corn- T.J. Mclntosh / Chemistry and Physics q[ Lipids 81 (1996} 117 13l 119 petition between the elastic energy of bending the 23 for eggPC and greater than 12 for eggPE. The interfaces and energies resulting from the con- water contents of lamellar phases of a variety of straints of interfacial separation'. They argue that PCs and PEs have been obtained by several tech- the propensity of a lipid system to form a non- niques, including NMR [38,39], differential scan- lamellar phase depends on its spontaneous radius ning calorimetry [40-43] and X-ray diffraction of curvature, which represents the minimum elas- [4,8,33,40,44-60]. As tabulated in a recent review tic free energy state of the lipid monolayer with [61], there is some variability in the results from respect to bend. Thus, the spontaneous radius of different laboratories and from lipids with differ- curvature represents the radius of surface that ent hydrocarbon compositions and chain linkages. these lipids would form in the absence of inter- However, from these many measurements, two monolayer packing constraints [32]. Evidence fa- general and important conclusions can be voring the spontaneous radius of curvature model reached: (1) for both PC and PE the water con- comes from the work of Leventis et al. [32] who tents are higher for liquid-crystalline than for gel analyzed a number of PE analogs with alkylated phases, and (2) PC absorbs more water than PE headgroups and Gruner et al. [33] who studied in either the liquid-crystalline or gel phase. For N-methylated PEs. Leventis et al. [32] found a example, in a particularly detailed and careful correlation between the hexagonal lattice repeat analysis, Nagle and Wiener [57] find that the dimension, which is a measure of the spontaneous maximum number of water molecules per lipid radius of curvature, and the lamellar-to-hexagonal molecule are: 14 and 23 for gel and liquid-crys- phase transition temperature of these lipids and talline phase dipalmit oylphosphatidylcholine Gruner et al. [33] found that both the hexagonal (DPPC), respectively, and 6 and 9 fl)r gel and lattice repeat dimension and the stability of the liquid-crystalline phase dilauroylphos- lamellar phase increase with increasing headgroup phatidylethanolamine (DLPE), respectively. methylation. Epand and Epand [34] have used A number of investigations have probed the titration calorimetry to measure the heats of reac- reasons for the differences in hydration of gel and tion between bilayers and lysoPC (which has a liquid-crystalline PEs and PCs. X-Ray diffraction spontaneous curvature opposite to that of phos- analysis of the structural changes that occur at the pholipids that form inverted hexagonal phases) gel to liquid-crystalline phase transition have been and relate these heats to the bilayer curvature particularly helpful. In the case of DLPE, it has strain. been found that although the area per lipid molecule increases from about 40 A 2 to about 50 A2 upon melting, the width of the fluid space 3. Hydration of lamellar and hexagonal phases between bilayers remains nearly the same in the gel and liquid-crystalline phase (about 5 /k, using In the lamellar phase, it has been known for a the definition of fluid spacing as the distance number of years that the hydration properties of between the physical edges of apposing bilayers) PC and PE bilayers are quite different. Adsorp- [8]. Other PEs also have about this same fluid tion isotherms have been obtained for both gel separation in the liquid-crystalline phase [62,63]. [35] and liquid-crystalline [36] lamellar phases of Therefore, in the case of PE the relatively small PC and PE. For both phases, oriented PC multi- increase in water content upon melting is due layers exposed to controlled humidity atmo- almost entirely to a change in area per lipid spheres take up more water than do PE molecule, and the resulting increase in the volume multilayers. In terms of lipid dispersions, early of the interbilayer fluid spacing. In the case of PC, NMR measurements by Finer and Darke [37] both the area per molecule and the width of the showed that lamellar PC dispersions take up sig- fluid spacing increase upon melting from the gel nificantly more water than do dispersions of PE; a to the liquid-crystalline phase [53,57]. Specifically, bulk water phase is present when the number of upon melting the area of DPPC increases from 48 water molecules per lipid molecules is greater than to 63 A 2 [64] and the fluid spacing between DPPC 120 T.J. Mclntosh / Chemistl3' am/Physics O~ Lipi& M (1996) 117 131 bilayers increases from about 12 /~ in the gel stress/X-ray diffraction experiments. In this os- phase [53] to about 18 A in the liquid-crystalline motic stress method [47.70 72] water is removed phase [64]. The fluid separation in the liquid-crys- from the macromolecular assembly (such as mul- talline phase of PC depends on the composition of tilamellar lipid bilayers or lipid hexagonal phases) the lipid hydrocarbon chains, varying from about by the application of known osmotic pressures. 15 A for EPC [53] to over 20 A for polyunsatu- The osmotic pressures are applied either by incu- rated diarachidonylphosphatidylcholine (DAPC) bating the lipid specimens in solutions containing [65]. Thus, in the case of PCs, the volume of the known concentrations of large neutral polymers fluid space and the number of water molecules per such as dextran or by equilibrating the sample lipid increase on going from the gel to the liquid- with a vapor of known relative humidity [47,72]. crystalline phases because of increases in both the For each pressure the distance between repeating area per lipid molecule and the width of the fluid units in the assembly is precisely measured by space between apposing bilayers. X-ray diffraction methods. At equilibrium in ex- The reasons for the differences in widths of the cess water, the distance between apposing repeat- interbilayer fluid spaces for PE and PC will be ing units is determined by the balance between the analyzed in detail in the next section. In terms of total repulsive pressure (P,.) and the total attrac- the differences in area per molecule between PE tive pressure (P~,), which for PC bilayers is the van and PC bilayers, two factors have been raised der Waals pressure [73]. When an osmotic pres- differences in volumes of the lipid headgroups and sure (P) is applied, then the spacing is set by the role of hydrogen-bonding. Dilatometry [26] P+ P~, = P,.. However, for pressures P > > P~, shows that the volumes of the PC and PE head- then P ~ P,., and plots of the known osmotic groups are 344 and 246 A ~, respectively, which pressure P versus the distance between repeating would allow a tighter packing of lipid molecules units provides the distance dependence of the in PEs, particularly in the gel phase [66]. Tran- total repulsive pressure Pr. The result is a pres- sient hydrogen bonds between the nitrogen and sure-distance or pressure-water volume relation phosphate groups on adjacent PE molecules are that can be integrated to determine the work of thought to inhibit lateral expansion of the bilayer dehydration. [25,67,68], which would help account for the Pressure-distance relations for bilayer phases smaller molecular area for PE in the liquid-crys- can also be measured directly with the surface talline phase. force apparatus, pioneered by Israelachvili and Fewer measurements have been made of the colleagues [74 77]. With this apparatus interac- water content of hexagonal phases. However, it tion forces are obtained for bilayers coated onto appears that hexagonal phases take up larger smooth mica surfaces by the use of interchange- volumes of water per lipid molecule than do able springs, with the intersurface separations lamellar phases [11,69]. For example, at 22°C the measured by an optical technique involving multi- hexagonal phase of DOPE takes up about 18 ple beam interference fringes. Similar pressure-dis- waters per lipid beyond which an excess water tance relations for specific phospholipids have phase forms [11]. In contrast, as noted above, been obtained with the osmotic stress technique lamellar phases of liquid-crystalline PEs take up and the surface force apparatus [78,79]. The between nine and 12 water molecules per lipid. agreement is particularly close for gel phase bilayers, and differences observed for liquid-crystalline bilayers could be due to the damping of entropic 4. Energetics of phospholipid hydration undulation pressures by the mica support in bilayers in the surface force apparatus [79]. 4.1. Methods As summarized in several recent reviews [72,80 83], pressure-distance relations have been The energetics of dehydrating an ordered array obtained for lipid lamellar phases by both the of macromolecules can be determined by osmotic osmotic stress and surface force apparatus meth- T.J. Mclntosh / Chemistry and Physics of Lipids 81 (1996) 117 -131 121 ods and for hexagonal phases by the osmotic and exhibits a definite downward bend at lower stress method. This review focuses on the en- applied pressures. ergetic factors involved in dehydrating lamellar and hexagonal phospholipid phases formed from 4.2. EneJigetics of hydration o/ PE hexagonal PC and PE. phases Fig. I compares typical osmotic stress data from the hexagonal phase of dioleoylphos- Considerable attention has been given to under- phatidylethanolamine (DOPE) and from the standing the physical origins of the repulsive inter- lamellar phase formed from natural PC isolated actions involved in hydrating (or dehydrating) from egg yolks (EPC). Similar comparisons, pre- both the hexagonal and lamellar phases. As noted sented in terms of pressure versus volume of by Rand et al. [69], in terms of water affinity the water, have been made for the lamellar and distinguishing feature of the two phases is the hexagonal phases of DOPE recorded at 14 and different geometries of the two phases. For the 22°C, respectively by Gawrisch et al. [84]. As seen hexagonal phase, a number of l:actors have been in Fig. 1, for both the lamellar and hexagonal considered in analyses of the observed repulsive phases, the total repulsive pressure (P) decreases pressure-distance relations [12,69.84,90], including monotonically with increasing repeat period. energies to bend the lipid monolayers that com- However, the shapes of the pressure-distance rela- prise the lipid cylinders, to hydrate the polar lipid tions from the two phases are quite different. For headgroups, to laterally compress the lipid chains, the lamellar phase, the log (P) versus repeat pe- and to fill the voids in the lipid hydrocarbon riod data points fall very near a straight line. This interstices. indicates that for most of the pressure range the P In terms of the bending energy, as an hexagonal decays exponentially with increasing separation phase is dehydrated by osmotic stress (Fig, I), the with a decay length of about I-2 A, a result that size of the water cavity decreases and the surround- has been found for a variety of phospholipid ing lipid monolayer must bend. As the monolayer bilayers [51,53,58,72,73,81,85 89]. In contrast, the bends, the areas of the polar and hydrocarbon ends pressure-repeat period data from the hexagonal of" the phospholipid molecule change in opposite phase decays more gradually with increasing re- directions as the molecule pivots around a position peal period at high applied pressures (log P > 7), of constant area [69]. That is, upon dehydration of the hexagonal I1 phase there is an increase in molecular area in the hydrocarbon region, but a I0 ...... decrease in molecular area in the polar region. The position of the pivotal position of constant area % along the lipid molecule has been analyzed by both E X-ray difl'raction [69] and NMR [91]. Kirk et al. ~-. [21] showed that variations in the radius of curva- e~ © ture (r) of the lipid tubes cause a change in the 6 [ GC % monolayer curvature Free energy of the fi~rm I • DOPE Hexagonal Phase c:~ AG=(K,/2)'(I/r--l:r.) ~ (1) [' EPC Lamellar Phase i "' 4 ...... c'c~ • where A G is the change in free energy per unit 30 40 50 60 70 Bragg Spacing (~) area, K~, is the monolayer bending modulus, and ro is the spontaneous radius of curvature of the Fig. 1. Osmotic stress experiments for DOPE in the hexagonal lipid layer. Several studies [12,69,84,90] have phase and EPC in the lamellar phase. Data are plotted as the shown that the pressure-distance data for the logarithm of applied osmotic pressure (log P) versus the observed Bragg diffraction spacings for the hexagonal and hexagonal phase can be approximated quite lamellar phases. Data are taken from Parsegian et al. [47] and closely by the energy of bending the lipid mono- Rand e1 a{. [69]. layer as given by the formalism of Eq. (l). In 122 T.J. Mclntosh / Chemistry attd Physics ~?/ Lipids 81 (I 996) 117 131 particular, Rand et al. [69] found an excellent agreement to the measured data over most of the l °,L measured pressure range when they defined the radius of curvature (r) as the distance from the wo -2 8 i center of the water cylinder to the constant-area e- per molecule pivotal position along the phospho- e~ lipid molecule. Although the measured pressure- distance curves could be fit quite well with this quadratic bending energy (Eq. (1)), Rand et al. ° o o [69] note that this does not exclude the possibility 45 50 55 60 65 of other kinds of interactions, especially at low Repeat Period (A) water contents where there are deviations between Fig. 2. Logarithm of applied osmotic pressure (log P) versus the observed data and the predictions of Eq. (1). lamellar repeat period for EPC bilayers. Data are from Parse- In terms of the hydration properties of lamellar gian et al. [47] (O) and Mclntosh et al. [97] (O1. and hexagonal phases, dioleoylphosphatidylethanolamine (DOPE) is a particularly nal phase transition in soy PE. In a similar man- interesting system. Above 25°C, DOPE forms an ner, Epand and Bryszewska [93] showed for several PEs that several salts affect the lamellar- hexagonal phase over a wide hydration range. to-hexagonal phase transition, which they argued However, at 15°C DOPE undergoes an unusual is due to changes in solvation of the lipid. More- hexagonal-to-lamellar-to-hexagonal phase transition sequence as water is removed [11,12,84]. A over, infrared spectroscopy experiments show a weakening of the hydrogen-bonded water shell at thorough analysis by Kozlov et al. [12] of the the lamellar-to-hexagonal phase transition in energetics of these transitions, as well as the usual eggPE [94] and fluorescence studies, using water temperature driven lamellar-to-hexagonal transi- soluble and lipid soluble fluorescent probes, indi- tion at full hydration, shows the important role cate that both the lipid dynamics and the lipid played by the bending energy (Eq. (1)). Moreover, headgroup hydration change at the lamellar-to- this study also points to contributions of other hexagonal phase transition [95]. energies, namely the hydration energy due to the affinity of the lipid polar groups for solvent and 4.3. Energetics of hydration of PC and PE an interstitial energy associated with the removal lamellar phases of voids from the hexagonal phase. For example, Kozlov et al. [12] find that in excess water, the Osmotic stress experiments provide critical in- lamellar-to-hexagonal phase transition induced by formation concerning the energetics of bilayer increasing temperature above 25°C depends criti- hydration. We first consider the case of PC bilay- cally on the magnitudes of the competing unbend- ers, which have been the most thoroughly studied. ing and interstitial energies. However, upon Fig. 2 shows pressure versus lamellar repeat dehydration at a constant temperature of 15°C, period data for EPC from two laboratories the lamellar-to-hexagonal phase transition de- [53,73]. The raw data are quite similar. The lamel- pends on the bending and interstitial energies, as lar repeating unit contains one lipid bilayer and well as a critical contribution from the energy of the fluid space between apposing bilayers. To hydration of the polar headgroups. determine the width of both the bilayer and fluid The role of headgroup hydration properties in space at each value of applied pressure, two meth- the relative stability of lamellar and hexagonal ods have been used, a gravimetric method [73,96] phases has also been indicated by other studies. and electron density profiles calculated by Fourier Indirect evidence for the role of water organiza- analysis [53,85,97]. These methodologies have tion comes from the work of Yeagle and Sen [92], been described in detail in recent reviews who found that the addition of chaotropic agents [72,81,83]. In the following section, the results of raises the temperature of the lamellar-to-hexago- the Fourier analysis are used. T.J. Mclntosh , Ctlemi.*'try and Phv,sics" ol Lipid,s' 81 (1996) / / 7 13/ 123

Electron density profiles have been calculated 1() • I)pl'C Suhecl for EPC bilayers for the entire range of applied !;! :o, ~ H,c osmotic pressures shown in Fig. 2 [53,97]. Fig. 3 • ~ • DAP(" shows typical profiles for EPC multilayers in excess water (no osmotic stress) and in 60% polyvinylpyrrolidone (PVP) solution (osmotic i pressure P = 5.7 × 107 dyn/cm ~, log P = 7.8). In Fig. 3, two unit cells are shown containing two apposing bilayers and the fluid space between bilayers. For each profile, the bilayer on the left is centered at the origin, so that the low density trough at 0 A corresponds to the terminal methyl O 5 I¢) 15 2t 2S Distance Between Bilayers (/~) groups in the bilayer center. The high density peaks centered near _+ 20 A correspond to the Vig, 4. l,ogarithm of applied osmotic pressure (log P) versus lamellar repeat period for subgel DPP(', EPC. and DAPC EPC headgroups, and the medium density regions bila3,ers. The equilibrium Ituid spacings in the absence of between the headgroup peaks correspond to the applied pressure are shown on the x-axis. Data are taken fiom methylene chains. The profiles of the bilayers on [65,89.97], the left nearly superimpose for samples in the presence and absence of applied pressure, indicat- osmotic pressure (dotted line, Fig. 3), showing ing that the bilayer thickness is not appreciably that the bilayers are squeezed together by this changed by this magnitude of osmotic pressure applied pressure. Profiles such as these can be [53,97]. This implies that the applied osmotic pres- used to estimate the distance between apposing sure is removing water primarily from the fluid bilayers at each applied pressure [53]. spacing between adjacent bilayers, and not from The total pressure-versus distance relations the bilayer headgroup region [53,97]. The head- shown in Fig. 2 depend on several non-specific group peaks from the adjacent fully hydrated interactions that have either been proposed theo- EPC bilayers are located at approximately 44 /k retically or demonstrated experimentally for elec- and 82 ,K. (solid line. Fig. 4). The medium density trically neutral phospholipid bilayers. These region between bilayers (centered at about 32 A) interactions include the attractive van der Waals corresponds to the middle of the fluid space be- pressure [98,99], the repulsive hydration pressure tween adjacent bilayers. This fluid space is consid- [72,73,100], and repulsive steric (entropic) pres- erably smaller for the bilayers subjected to sures due to headgroup movement [85,97], molecular protrusions [101,102], and bilayer I:it g P(" hi "~, atel Egg P(" in 61)91 PVP undulations [103,104]. In the past few years a >.G" number of experiments have been performed to obtain information on the relative contributions of each of these pressures for PC bilayers [65.89,105]. One set of experiments compared the total pressure-distance relations for PC bilayers in different phases, which would be expected to have different

al -20 20 40 60 ~l, 100 contributions fi'om entropic pressures [89]. In the Distance from Bilayer Center (A) liquid crystalline phase the hydrocarbon chains are fluid, in the gel phase the chains have rota- Fig. 3. Electron density profiles for EPC in water and in 60V,, PVP solution. In each profile two unit cells are shown, includ- tional motion but are rigid in the sense of posi- ing two bilayers and the interbilayerfluid space. Data are from tional order, and in the subgel phase the lipid acyl Mclntosh and Simon [53]. chains are crystallized in the phme of the bilayer 124 T.J. Mclntosh / Chemistry and Physics o! Lipi&" 81 (1996) 117 I.~1

[49,50]. Since the bending stiffness is much larger extending from df ~ 4-8 ,~ is primarily a hydra- for gel [106] (or subgel) bilayers compared to tion pressure, arising from the orientation of wa- liquid crystalline bilayers, the magnitude of the ter by the polar headgroups. Other experiments undulation pressure [104,107] should be much [60,86,88,110] are also consistent with the pres- lower in the gel and subgel phases [89]. Also, since ence of a large hydration pressure in this distance the cohesive van der Waals energy in the hydro- range. carbon core of the bilayer should be significantly The biggest difference among the three sets of larger for gel or subgel bilayers than for liquid data in Fig. 4 is that the repulsive pressure has the crystalline bilayers [67,108], molecular protrusions shortest range for subgel DPPC and the longest should be smaller in the gel and subgel phases range for DAPC, such that equilibrium fluid sepa- [89]. Therefore, McIntosh and Simon [89] argue rations are about 8 A for subgel DPPC [89], 15 A that the effects of lipid protrusions and bilayer for EPC [53], and 20 ,~ for DAPC [53]. These undulations should be quite small in the subgel differences can be explained by increases in the phase compared to the liquid crystalline phase. undulation pressure due to the differences in the McIntosh et al. [65] also analyzed bilayers com- bilayer bending moduli for the three systems; it posed of polyunsaturated diarachidonoylphos- has been found [65] that the measured equilibrium phatidylcholine (DAPC) bilayers, which have fluid spacings (Fig. 4) are quite close to those much smaller bending moduli than EPC bilayers predicted by the theoretical treatment of undula- [109]. Therefore, compared to EPC bilayers, the tions developed by Evans [111]. Thus, thermal undulation pressure is expected to be smaller for undulations can explain the relatively large fluid subgel DPPC and larger for DAPC bilayers separations observed for DAPC. [65,89]. Fig. 4 shows pressure-distance data for These data, along with experiments determining bilayers of subgel DPPC, liquid crystalline EPC, the temperature dependence of the pressure-dis- and DAPC, where the distance between bilayers is tance relations [105] indicate that both hydration estimated from electron density profiles such as repulsion and entropic repulsive pressures con- shown in Fig. 3. At interbilayer spacings less than tribute to the total repulsive pressure between PC about 4 A, the total repulsive pressure is largest bilayers, in agreement with theoretical treatments for subgel DPPC and smallest for DAPC. For [104,107,112,113]. these small interbilayer separations the dominant Pressure-distance relations have also been pressure is thought to be a short-range steric obtained for PE bilayers [53,58,72]. Fig. 5 pressure between headgroups from apposing bi- shows typical pressure-distance relations for layers [65,85,89,97]. The magnitude of such a liquid-crystalline 1-palmitoyl-2-oleoylphosphati- steric pressure should depend on the density of PC headgroups at the interface [85~97]. Therefore, 9[ .... i ~ ~ • SOPC ) as observed experimentally (Fig. 4), for these ! ¢ pOPE j' small spacings DAPC (which has the largest area per molecule) should have the smallest steric pressure and subgel DPPC (which has the smallest ! area per molecule) should have the largest repul- 61 ~ • . sive pressure. The pressure-distance curves are similar for the liquid crystalline and subgel phases for 4 A < dr < 8 A (Fig. 4), indicating that for this region 5O 55 60 65 the same interactions are responsible for the re- Repeat Period (~) pulsive pressure [89]. Since undulations and pro- Fig. 5. Logarithm of applied osmotic pressure (log P) versus trusions should be markedly damped in the subgel lamellar repeat period for SOPC and POPE bilayers. The phase compared to the liquid-crystalline phase, repeat periods in the absence of applied pressure are shown on McIntosh and Simon [89] argue that the pressure the .v-axis. Data are taken from Rand et al. [58]. T.J. Mclntosh /' Chemistry and Physics of Lipid~ 81 (1996) 117 131 125

68 dylethanolamine (POPE) and 1-stearoyl-2-oleoyl- phosphatidylcholine (SOPC) bilayers. Since the hydrocarbon chain compositions of these two °g 64 lipids are similar (POPE has two more CH2 o [ groups than SOPC), their bilayer thicknesses are e~5 ~,0! quite similar [58]. Therefore, the observed differ- } [ • ences in repeat periods are due primarily to differ- e~ 56 ences in the thicknesses of the interbilayer fluid spacings. From Fig. 5 it can be seen that the 52[ ..... range of the total pressure is much larger for Mol Fraction SOPC SOPC than for POPE. This can be appreciated Fig. 6. Lamellar repeat period of" POPE SOPC bilayers ill most easily by noticing that the repeat periods of excess water. Data are taken fiom Rand et al. [58]. the fully hydrated lipids in the absence of applied pressure (data points displayed on the x-axis) are hydration properties to those of PC. They also 64.6 A for SOPC and 53.2 /k for POPE [58]. As observed that, when mixed with POPE, SOPC the multilayers are squeezed together by increas- causes disproportionate increases in hydration. ing osmotic pressure (P) the repeat period and the For example, Fig. 6 shows that relatively small interbilayer fluid spacing change by a much larger amounts of SOPC increase the lamellar repeat amount for SOPC. Thus, at an applied pressure period (and thus the fluid spacing) of POPE bilay- of approximately 3.2 x 10v dyn/cm2 (log P ~ 7.5) ers. Rand et al. [58J argue that these data can be the lamellar repeat period decreases by less than 2 explained by variable contributions from attrac- /k for POPE, but over 10 A for SOPC, Similar tive and repulsive hydration pressures. results for other liquid-crystalline PC and PE To obtain further information on the short- bilayers have been obtained by the osmotic stress range interactions between bilayers Mclntosh and method [53] and for gel PC and PE bilayers with Simon [116] measured pressure-distance relations the surface force apparatus [114]. for PC and PE bilayers swollen apart with known There have been several proposals to explain electrostatic repulsive pressures. In these experi- the observed differences in the pressure-distance ments the interbilayer distance was extended by relations for PC and PE bilayers (Fig. 5) and the incorporating into both PE and PC bilayers vari- smaller equilibrium fluid separations in PE bilayers (see above). For example, compared to PC ous concentrations of the negatively charged lipid bilayers, it has been argued that PE bilayers have: phosphatidic acid (PA). The rationale is that if an a stronger van der Waals attractive pressure [51], additional attractive interaction exists between PE a smaller repulsive hydration pressure [33], less bilayers, then it should take a larger repulsive dynamical freedom in the PE headgroup due to electrostatic pressure (the addition of more nega- intrabilayer hydrogen bonds [115], an attractive tively charged lipid) to swell PE bilayers than PC hydration pressure [58], or a strong, short-range bilayers. In strong support of this idea is the work interaction due to either H-bonding water bridges of Van der Kleij et al. [117], who found that the between PE bilayers [8,30,116] or electrostatic in- lamellar repeat period of PE:PA mixtures remains teractions between the amine and phosphate virtually constant for 0-4 mol% PA whereas the groups on apposing bilayers [8,115,116]. repeat period of PC bilayers increases significantly Rand et al. [58] performed two types of experi- with the incorporation of 4 tool% PA. To simplify ments to analyze the differences in the pressure- the analysis, Mclntosh and Simon [116] used distance behavior of PCs and PEs. First, they gel phase dipalmitoylphosphatidylethanolamine determined the effects of successive methylations (DPPE) and dipalmitoylphosphatiditic acid of the PE headgroup, and second they examined (DPPA) bilayers, where the protrusion and undu- mixtures of PE and PC. They found that a single lation pressures are negligible [81,89]. Fig. 7 methylation of the PE headgroup converts its shows pressure-distance data for DPPE and 19:1 126 T.J. Mchttosh , CYtemisto' and Physic.~ ql LilmA S I f 1990) 117 Id I and 4:1 molar mixtures of DPPE/DPPA. As is the area of E ~ - I).7 erg/cm -~, which is much larger case for DOPE bilayers (Fig. 5), applied osmotic than the adhesion energy of -0.15 erg/cm 2 for pressures decrease the lamellar repeat period and DPPC obtained with the surface tbrce apparatus interbilayer fluid spacing of DPPE by only 2 [114]. The attractive van der Waals energy be- (Fig. 7). The addition of 20 tool% DPPA increases tween DPPE bilayers is calculated to be about the repeat period so that the pressure-distance - 0.03 erg/cm ~ [l 16]. Thus, for DPPE bilayers the data fall near the pressure expected from electro- adhesion energy is much larger in magnitude than static repulsion, shown as the solid line in Fig. 7. the van der Waals energy. The adhesion energy However, the addition of 5 tool% DPPA does not between bilayers (E) can be written E = E, + E,., change the pressure-distance curve at all (Fig. 7). where E, and E, represent the attractive and That is, the interbilayer spacing is the same for repulsive energies, respectively, at the equilibrium 19:1 DPPE/DPPA and DPPE. In contrast, in the fluid separation. Since the repulsive component of case of DPPC, the addition of 5, 10, or 20 tool% the energy (E,.) must be positive, the only way this DPPA swells (disjoins) the bilayers in a systematic energy equation can be balanced for DPPE is for manner predictable from double-layer theory there to be an additional attractive pressure be- [116]. Stated another way, the addition of increas- sides the van der Waals pressure. In contrast, for ing concentrations of charged lipids causes PC DPPC it has previously been shown that this bilayers to swell or disjoin continuously, whereas energy equation can be balanced by considering PE bilayers disjoin only when a critical concentra- only the repulsive hydration pressure and the tion of charged lipid is added. attractive van der Waals pressure [89]. These experiments with DPPE/DPPA mixtures These experiments (Fig. 7) indicate that the provide information on the adhesion energy for smaller interbilayer spacing ['or PE compared to PE bilayers, which is critical Ibr understanding PC cannot be due to smaller repulsive pressures, the hydration properties of PE. The results of but must be the result o1" an additional strong [116] indicate that the introduction of about 10 attractive interaction for PE bilayers [116]. Attrac- tool% DPPA adds just enough charge to disjoin tive interactions that have been proposed for PE the DPPE bilayers. Therefore, the electrostatic bilayers involve mterbilayer interactions between pressure of 9:1 DPPE/DPPA is the minimum the N4H~ group in one bilayer and the PO 4 pressure necessary to disjoin DPPE bilayers. Inte- group in the apposing bilayers, either through gration of the pressure-distance curve for 9:1 electrostatic interactions [4,8,115], direct hydro- DPPE/DPPA gives the adhesion energy per unit gen-bonding [4], or hydrogen-bonded water bridges [8,58]. As detailed in [1161, such interac- 8 ...... ' ' r , ~ , tions are more probable for PE bilayers than PC ~. ! ~ DPPE I 19:1 DpPE:DPPA bilayers for several reasons. In terms of electrostatic interactions, the headgroup motions of hydrated PC bilayers are greater than in hydrated ¢-- 6 "'-. PE bilayers [118], and. since the trimethylammo- nium moiety o1" PC has a greater volume than the O0 O-.~ O amine of PE, the positive charge of the PE head- -\ 4 ~, group is more localized in the plane of the bilayer. In lerms of hydrogen-bonding, recent molecular dynamics simulations [115,119] show that water 20 4(} 60 8t) 100 Distance Between Bilayers (A) molecules are strongly hydrogen-bonded to the PE headgroups [115,119], whereas water forms a Fig. 7. Logarithm of applied osmotic pressure (log Pt versus clathrate-like structure around the PC headgroup distance between bilayers for DPPE contaMng 0, 5, and 20 tool% DPPA. The solid line represents the predicted electro- [115,120]. Moreover, in support of the idea of static repulsive pressure for 4:1 DPPE/DPPA. See text for hydrogen-bond water bridges, electron density details. Data are taken from Mclntosh and Simon [116]. profiles [8,53,116] show that the interbilayer fluid T.J. Mclntosh Chemistry and Physics of Lipi& 81 (1996) 117-131 127 spacing in PE multilayers is so narrow that it [4] J.M. Seddon, G. Cevc, R.D. Kaye and D. Marsh (19841 could be spanned by one or two water molecules X-Ray diffraction study of the polymorphism of hydrated diacyl- and dialkylphosphatidylethanolamines. hydrogen-bonded to the amine and phosphate Biochemistry 23. 2634 2644. groups from apposing bilayers. [5] E. Shyamsunder, S.M. Gruner, M.W. Tare, D.C, As noted above, a major difference between PC Turner, P.T.C. So and C.P.S. Tilcock (1988) Observa- and PE bilayers is that the fluid separation in fully tion of inverted cubic phase in hydrated DOPE mem- hydrated bilayers is the same for gel and liquid branes. Biochemistry 27, 2332 2336. [6] R.N.A.H. Lewis, D.A, Mannock, R.N. McElhaney, crystalline phase PEs [8], whereas the equilibrium D.C. Turner and S.M. Gruner (1989) Effect of fatty acyl fluid separation is considerably larger for the liq- chain length and structure on the lamellar gel to liquid- uid crystalline phase compared to the gel phase crystalline and lamellar to reversed hexagonal phase for PC bilayers [53,81]. As shown by the data in transitions of aqueous phosphatidylethanolamine disper- Fig. 4, liquid crystalline PC bilayers swell to a sions. Biochemistry 28, 541 548. greater extent than gel or subgel phase PC bilay- [7] R.N.A.H. Lewis and R.N. McEIhaney (1993) Calorimet- ric and spectroscopic studies of the polymorphic phase ers primarily due to the presence of the repulsive behavior of a homologous series of n-saturated 1,2-dia- undulation pressure in liquid crystalline bilayers cyl phosphalidylethanolamines. Biophys. J. 64, 1081 [89]. Since the area compressibility moduli are 1096, similar for liquid crystalline PC and PE bilayers [8] T.J. Mclntosh and S.A. Simon (19861 Area per molecule [106] and the bilayer thicknesses are comparable and distribution of water in fully hydrated dilau- roy[phosphatidylethanolamine bilayers. Biochemistry 25, [8,53], the bending moduli and repulsive undula- 4948 4952. tion pressures should be comparable for liquid [9] B.D, Ladbrooke and D. Chapman (19691 Thermal anal- crystalline PC and PE bilayers. However, in the ysis of lipids, proteins, and biological membranes. A case of PE bilayers, McIntosh and Simon [116] review and summary of some recent studies. Chem. argue that attractive interactions, probably due to Phys. Lipids 3, 304 367. H-bond water bridges, overcome the undulation [10] R.M. Epand ([990) Hydrogen bonding and the ther- motropic transitions of phosphatidylethanolamines. pressure and prevent liquid crystalline PE bilayers Chem. Phys, Lipids 52, 227 230. from swelling. [11] R.P. Rand and N.E. Fuller (19941 Structural dimensions and their changes in a reentrant hexagonal-lametlar transition of phospholipids. Biophys. J. 66, 2127 2138. [12] M.M. Kozlov, S. Leikin and R.P. Rand 119941 Bending. Acknowledgements hydration and void energies quantitatively account ~\)r the hexagonal-lamellar-hexagonal reentrant phase transition in dioleoylphosphatidylethanolamine. Biophys. J. 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