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* Department of Biological Sciences, Brock University, St. Catharines, Ontario, Canada L2S 3A1; and Departamento de Física Geral, Instituto de Física, Universidade de São Paulo, São Paulo, Brazil
Correspondence: Address reprint requests to Nola L. Fuller, 500 Glenridge Ave., St. Catherines, Ontario, Canada L2S 3A1. Tel.: 1-905-688-5550-4166; Fax: 1-905-688-1855; E-mail: nfuller{at}brocku.ca.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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4.0, DOPS (with no td) adopted the HII phase on its own, in agreement with previous results, suggesting a reversal in curvature upon protonation of the serine headgroup. In contrast, when td was present, DOPS/td formed a lamellar phase of limited swelling whose dimension increased with pH. DOPS/DOPE/td mixtures formed HII phases whose dimension increased both with pH and with DOPS content. With tetradecane, estimates put 1/R0p for DOPS at pH 2.1 at zero. Tetradecane apparently affects the degree of dissociation of DOPS at low pH. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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) and activation of various phospholipases (Buckland and Wilton, 2000), protein kinase C (Nishizuka, 1984), and components of the blood coagulation process (Lentz, 1999). In addition, PS interacts with other components of the mammalian membrane such as sterols, cholesterol being the most important representative. Phospholipid, when hydrated, can aggregate in several different lyotropic phases. Increasingly, evidence suggests that the tendency of some specific phospholipids to form phases other than bilayers may have implications for biological functions (Chernomordik et al., 1995b; Cullis et al., 1985). The most common phases formed by naturally occurring phospholipids are the bilayer lamellar (L
) phase and the reverse hexagonal phase (HII) (Chen and Rand, 1997; Epand, 1997, Rand and Fuller, 1994; Seddon, 1990). Lipids that form the most common of the nonlamellar phases, the HII phase, appear to be of fundamental relevance for many processes carried out by biomembranes (de Kruiff, 1987; Seddon, 1990). These lipids, incorporated within bilayers, appear to produce packing stresses that affect both membrane protein conformation and topological changes required in processes like membrane fusion and fission. Most recently, in engineered membranes for the application of drug and DNA delivery, the tendency of the lipid components to adopt the hexagonal phase was observed to correlate with fusion or transfection efficiency (Koltover et al., 1998; May et al., 2000).
The structural preference of lipid mixtures for the lamellar or hexagonal phase is dictated by the minimization of molecular free energies of the component molecules (May and Ben-Shaul, 1999). Remarkably, the total energy difference between the L and HII geometries can be extremely small (Kozlov et al., 1994b). Although some internal individual molecular constants contributing to the free energy of different lipid molecules cannot be measured directly (May and Ben-Shaul, 1999), the elastic constants of the lipid ensemble, monolayer spontaneous curvature (c0) and bending modulus (Kcp), can be determined and give some indication of phase preference. By convention, large negative curvature lipids tend to form HII phases, large positive curvature lipids form micelles or HI phases, and small curvature lipids form bilayers.
The structure and phase behavior of phosphatidylserines has been studied by several techniques (Browning and Seelig, 1980; de Kroon et al., 1990; Hope and Cullis, 1980; Portis et al., 1979). These studies have shown that at physiological or neutral pH, PS forms a lamellar phase upon hydration, and is capable of stabilizing nonbilayer forming lipids in a bilayer structure (Cullis and Verkleij, 1979). Thermodynamic properties of the negatively charged PS bilayers are not very different from zwitterionic phospholipids. Chain melting transition temperatures for pure diacyl PSs are only slightly higher than for the corresponding PCs, and lower than PEs. Transition enthalpies were found to be slightly larger. 2H and 31P NMR studies further show that the fatty acyl chain region of the PS bilayer is quantitatively similar to PC. In contrast, however, the headgroup order is more rigid than that of other phospholipids (Browning and Seelig, 1980), likely due to intermolecular electrostatic interactions (Roux et al., 1989) or hydrogen bonding (Boggs, 1987).
Under certain conditions, bilayer PS has been reported to undergo a transition to nonbilayer phases. Specifically, the HII phase has been observed in the anhydrous state (Hauser et al., 1982), in aqueous dispersions containing lithium ions (Cevc et al., 1985), and in aqueous dispersions at low pH (de Kroon et al., 1990; Hope and Cullis, 1980). This transition has led to the question of whether PS changes its curvature to highly negative upon neutralization of its charge. Significantly, in this specific case, Bezrukov et al. (1999) have shown that alamethicin channels change their gating properties in PS-containing membranes when the pH is changed. This is an explicit example of a protein's activity being modulated by the properties of its host lipid. We attempt here to measure some of these properties of PS. In this work we measure the spontaneous curvature of negatively charged dioleoylphosphatidylserine (DOPS) at neutral pH, and estimate the change in curvature at pH 2. A mixed lipid system, containing dioleoylphosphatidylethanolamine (DOPE) and various concentrations of DOPS, is used to give an HII phase. The radius of curvature of the equilibrium structure, in excess tetradecane (td) and excess water, is determined by x-ray diffraction. The contribution of DOPS to the negative curvature of DOPE/DOPS mixtures allows the spontaneous curvature of DOPS to be calculated. In addition, osmotic stress experiments (Leikin et al., 1996; Parsegian et al., 1986) are used to measure the effect of DOPS on the bending modulus of the DOPE monolayer.
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MATERIAL AND METHODS |
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Stock mixtures, each of different DOPE/DOPS composition, were prepared by mixing the lipids in chloroform solution, and then drying by rotary evaporation and vacuum desiccation. Unless otherwise stated, to relieve interstitial packing stresses, 16 wt% tetradecane was added to the dried lipid stocks and equilibrated as described previously (Leikin et al., 1996; Rand and Fuller, 1994; Rand et al., 1990). Aliquots from each stock were hydrated to varying degrees by either: 1), adding weighed amounts of distilled water; 2), adding excess amounts of PEG solutions of known osmotic pressure; or 3), adding excess amounts of various 100 mM buffer solutions at various pH values. The resulting equilibrated structures were investigated at 20°C by x-ray diffraction and their dimensions determined with a measuring error of ± 0.1 Å. Sample to sample variation including all experimental errors was a maximum of ± 0.5 Å. Thin-layer chromatography showed no degradation (<0.1%) of td-containing samples after equilibration. Degradation in td-free samples was <1%.
Structural analysis
The DOPE/DOPS/td lipid mixtures (studied here from 0 to 30 mol% DOPS) with added tetradecane gave single, well-ordered, inverted hexagonal (HII) phases at all levels of hydration. The HII phase has been well characterized (Epand, 1997; Fuller and Rand, 2001; Rand and Fuller, 1994; Seddon, 1990), and is shown schematically in Fig. 1. Following the method originally introduced by Luzzati (Luzzati and Husson, 1962), the hexagonal lattice is divided by a cylindrical surface (the Luzzati plane) into separate compartments containing lipid and water. In this way, at known volume fractions of water (
w) and lipid (1 - w), dimensions of the water cylinders and molecular dimensions of the lipid compartment can be determined (Eqs. 1–5, Fig. 1). We then follow a previously published procedure for determining structural and elastic characteristics of the HII phases.
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): the neutral plane (where bending and compression are energetically uncoupled) (Kozlov and Winterhalter, 1991a; Kozlov and Winterhalter, 1991b)), or the pivotal plane (where the molecular area remains constant on bending) (Rand et al., 1990). Here, we analyze the experimental data with reference to the pivotal plane.
Given the following geometric relation,
 | (6) |
Using this volume, now called Vp, giving the position of the pivotal plane, the radius of curvature at the pivotal plane (Rp) can be calculated using another geometric relation:
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Knowing the radius of curvature at the pivotal plane, Rp, the elastic free energy, F, of the hexagonal phase (normalized per phospholipid molecule) can be approximated by the energy of bending (Helfrich, 1973; Kirk et al., 1984).
 | (7) |
Finally, by determining the elastic parameters of the lipid mixtures under conditions of osmotic stress (Gruner et al., 1986; Rand et al., 1990), we relate the elastic energy given by Eq. 7 with the osmotic work done by osmotic stress (
):
 | (8) |
A plot of (Rp2) vs. (1/Rp) gives, from the slope, the monolayer bending modulus (Kcp) (Gruner et al., 1986; Rand et al., 1990).
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RESULTS |
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).
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Data analysis
Pivotal plane
Molecular dimensions, including Aw and Rw, were calculated using the equations described in Fig. 1 for all samples in less than excess water where the volume fraction of water was known. To standardize these calculations for different DOPE/DOPS/td mixtures, an effective molecule is described, which is one DOPE plus an appropriate fraction of DOPS and a fraction of tetradecane. Diagnostic plots were constructed (Fig. 4 A) using normalized areas and volumes. Eq 6 can be rewritten:
 | (9) |
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; Fuller and Rand, 2001; Leikin et al., 1996; Szule et al., 2002).
Spontaneous curvature and bending modulus
Knowing the position of the pivotal plane, the spontaneous radius of curvature (R0p) is calculated for each DOPE/DOPS/td mixture at the pivotal plane using Eq. 5 and the equilibrium conditions determined in Table 1. If the spontaneous curvature (1/R0p) of the mixtures is a linear function of DOPS molar fraction, then the apparent spontaneous radii of curvature for DOPS and DOPE can be determined from the best fit line to the data. This data is shown plotted in Fig. 4 B and yields: R0pDOPS = +144 Å and R0pDOPE = -30 Å.
Osmotic pressure data (Fig. 3 B) can now also be analyzed in terms of the radius of curvature at the pivotal plane. Fig. 5 A shows this data replotted as described in the Materials and Methods section. The slope of each line in the Rp2 vs. 1/Rp plot gives the bending modulus (Kcp) for each mixture. Fig. 5 B shows the bending modulus as a function of DOPS content. It is evident that DOPS has no significant effect on the monolayer bending modulus previously determined for DOPE (Leikin et al., 1996).
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) and NMR (de Kroon et al., 1990; Hope and Cullis, 1980) have shown that at low pH, fully hydrated pure DOPS with no tetradecane, forms an inverted hexagonal phase. These experiments were repeated as described in the methods, and the results confirmed in the present study (Fig. 6). From pH 1 to pH 4, an HII lattice, which is significantly smaller than that of DOPE, increases in spacing from 
56 Å to 65 Å, and coexists with a small dimension (48 Å) lamellar phase at the lower pH values.
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95 Å at pH 2.1, to 140 Å at pH 3.2. In DOPS, tetradecane apparently reverses the effects of lowering pH. Fig. 7 shows the equilibrium dimensions of the tetradecane-containing hexagonal phases formed at low pH by DOPE/td, and by DOPS/DOPE/td mixtures containing 20, 25, and 30 mol% DOPS. The DOPE/DOPS/td mixtures all form HII phases that increase in dimension with pH. Unpredictably (because DOPS at low pH forms a smaller HII phase than DOPE), they are all larger than the HII phase formed by DOPE. As a first approximation, to get an estimate of R0p for DOPS under these low pH conditions, we use the equilibrium lattice dimension (large excess buffer reservoir) of the samples at pH 2, and calculate the volume fractions of lipid and water assuming lipid molecular dimension, dl, equal to that for the corresponding lattice dimension measured at neutral pH. Many previous measurements show that any changes in dl are very small compared to the changes we observe here in the repeat lattice dimension of the fully hydrated hexagonal phase. This can be seen in Table 1. Thus we estimate the spontaneous radius of curvature at the pivotal plane for DOPS in the same manner as described above. The results of these calculations are shown in Fig. 8. At pH 2, the observed dependence of curvature on DOPS concentration gives its apparent spontaneous radius of curvature as essentially infinite, i.e., flat. For comparison, using the same assumption for dl, the pure DOPS samples fully hydrated without tetradecane, equilibrate at an estimated radius of curvature of -23 Å at pH 2 (protonated), and at -50 Å in 1M NaCl (screened) (unpublished data). Again tetradecane apparently offsets the effects of protonation at low pH.
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DISCUSSION |
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), and in affecting the activity of membrane-bound and associated proteins (Bezrukov et al., 1999). In this work, we have attempted to assign quantitative values to some lipid elastic constants that describe the phosphatidylserine molecule DOPS.
We have extended previous studies using DOPE/DOPS mixtures, designed to measure the effect of surface charge on monolayer curvature (Lerche et al., 1992). In this system, both lipids have the same hydrocarbon chains, and when salt screened, PS headgroups have been shown to have similar hydration properties to PE (Rand and Parsegian, 1989). The observed increase in the radius of curvature at the water interface, with increasing PS concentrations, was attributed to charge alone, and was in agreement with theoretical predictions (Kozlov et al., 1992).
The data presented here agree with and extend the previous data for DOPE/DOPS mixtures. Further analysis, using the procedure originally described by Leikin et al. (1996), allow the calculation of the position of a pivotal plane within the monolayer where the molecular area does not change on bending. The elastic constants, surface area (A0p), spontaneous curvature (1/R0p), and bending modulus (Kcp) were measured for the DOPS molecule at this plane. All samples used in this analysis (from 0 to 30 mol% DOPS) were pure hexagonal phases containing 16 wt% tetradecane. Tetradecane reduces chain-packing stresses or interstitial energy, and allows the monolayers to relax to their spontaneous radius of curvature (Kozlov et al., 1994a; Rand et al., 1990; Rand and Parsegian, 1997). This is important in determining spontaneous curvature because without added tetradecane, in mixtures of 15 mol% DOPS and above, the lamellar phase is the lowest energy phase (Lerche et al., 1992).
The position of the monolayer pivotal plane was determined to be independent of DOPS concentration and not detectably different from that of pure DOPE. The diagnostic plots (Fig. 4 A) emphasize experimental scatter in the data, but no systematic differences corresponding to DOPS concentration could be found. A value of Vp/Vl = 0.3 for the pooled data agrees with all previous measurements (Chen and Rand, 1997; Fuller and Rand, 2001; Leikin et al., 1996; Szule et al., 2002) and corresponds to a position close to the hydrocarbon polar group interface (2 CH2 groups on the hydrocarbon side).
The apparent spontaneous radius of curvature for DOPS was determined to be +144 Å. This represents the first positive curvature reported for a diacyl phospholipid, and likely is largely due to electrostatic interactions of the charged headgroup. By contrast, the apparent spontaneous radius of curvature of the highly hydrated but neutral lipid, DOPC, has been measured to be from -143 Å to -200 Å (Szule et al., 2002). Positively curved, charged DOPS confined in a flat membrane would be expected to exert packing stresses opposite to those of negatively curved lipids and so have opposite effects on membrane deformations or integral protein conformational changes. Such effects have been reported for lysoPC, which has been measured to have a positive radius of curvature from +38 Å to +58 Å. For example, lysoPC is reported to inhibit or promote different stages of membrane fusion depending on its location in either the proximal or distal monolayer (Chernomordik et al., 1995a,b; Chernomordik et al., 1993), and its effect on gramicidin channels is seen to increase channel formation and duration (Lundbaek and Anderson, 1994).
Interestingly, as previously reported, pure DOPS (without added tetradecane) undergoes a dramatic change in structure from lamellar to hexagonal under certain conditions including low pH. These results were confirmed here, and suggest a switch in curvature from positive to negative. Using an estimate for lipid thickness based on the common dehydrated data measured for all mixtures, the radius of curvature at the pivotal plane for this hexagonal phase would be approximately -23 Å (even smaller than DOPE). This apparent reversal in curvature from positive to highly negative is likely due to protonation of the PS headgroup at low pH and is consistent with the estimated pK value of 4.2 ± 0.2 found by Kroon et al. (1990) for the carboxyl group. Protonation would reduce electrostatic repulsion, increase hydrogen bonding, and decrease hydration of the headgroup, possibly resulting in a different orientation. All of this would change the balance of the energies stemming from headgroup interactions, chain-packing interactions, and surface tension associated with the water interface. Under these conditions, minimization of the energy results in the formation of this small dimension hexagonal phase (sometimes in equilibrium with a dehydrated lamellar phase). Because the dimension of this hexagonal phase is even smaller than that of DOPE, chain-packing stresses are believed to be at a minimum.
Following our normal routine or protocol, to get a measure of the spontaneous (relaxed) radius of curvature of HII phase lipids, low pH samples of DOPS and DOPE/DOPS mixtures were examined, not only in excess aqueous buffer, but also containing excess hydrocarbon solvent (tetradecane). Surprisingly, DOPS and DOPE/DOPS mixtures behave very differently at low pH when tetradecane is added. At pH 2, fully hydrated DOPE/DOPS/td mixtures form pure hexagonal phases, but the dimension of these phases is dependent on DOPS content in a manner that gives an estimate for DOPS curvature that is essentially flat. Indeed, consistent with this, pure DOPS at pH 2 with added tetradecane forms a lamellar phase of large dimension (93 Å) but limited swelling. This highly hydrated lamellar phase (the PS bilayer is only 39 Å thick) implies effective electrostatic charge repulsion, and is in sharp contrast with the extremely small dimension hexagonal phase formed without tetradecane. Clearly tetradecane appears to reduce the protonation of DOPS at low pH. Although more studies are necessary to determine the role of tetradecane in these systems, one interesting possibility is that tetradecane interaction somehow allows the hexagonal lattice to expand and take up more water, and/or affect the degree of polar group ionization. Cevc et al. (1985) has suggested a similar effect of hydrocarbon expansion by high temperature.
The application of osmotic stress technique (Leikin et al., 1996; Parsegian et al., 1986) using the DOPE/DOPS/td mixtures allowed us to measure the effect of increasing concentrations of DOPS on the bending modulus of the DOPE monolayer in the hexagonal phase. No significant effect of DOPS on bending modulus could be measured from our data, suggesting that Kcp for DOPS in the hexagonal phase is not significantly different from DOPE at 11 kT. Thus, our data could not detect a theoretical prediction (Kozlov et al., 1992, Winterhalter and Helfrich, 1988) that surface charge would influence rigidity in the monolayer.
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ACKNOWLEDGEMENTS |
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Submitted on September 26, 2003; accepted for publication May 6, 2003.
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30%, 
25%, 
20%, 
15%, 
10%, • 8%, and 
0% DOPS. Horizontal lines at equilibrium dimensions were drawn using the relationship found in 
