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Formation of Cubic Phases from Large Unilamellar Vesicles of Dioleoylphosphatidylglycerol/Monoolein Membranes Induced by Low Concentrations of Ca2+ Tarek S. Awad,† Yoshihide Okamoto,‡ Shah Md. Masum,† and Masahito Yamazaki*,†,‡ Materials Science, Graduate School of Science and Engineering, Shizuoka University, 836 Oya, Shizuoka 422-8529, Japan, and Department of Physics, Faculty of Science, Shizuoka University, Shizuoka, 422-8529, Japan Received July 2, 2005. In Final Form: October 7, 2005 We developed a new method for the transformation of large unilamellar vesicles (LUVs) into the cubic phase. We found that the addition of low concentrations of Ca2+ to suspensions of multilamellar vesicles (MLVs) of membranes of monoolein (MO) and dioleoylphosphatidylglycerol (DOPG) mixtures (DOPG/MO) changed their LR phase to the cubic phases. For instance, the addition of 15-25 mM Ca2+ to 30%-DOPG/ 70%-MO-MLVs induced the Q229 phase, whereas the addition of g28 mM Ca2+ induced the Q224 phase. LUVs of DOPG/MO membranes containing g25 mol % DOPG were prepared easily. Low concentrations of Ca2+ transformed these LUVs in excess buffer into the Q224 or the Q229 phase, depending on the Ca2+ concentration. For example, 15 and 50 mM Ca2+ induced the Q224 and Q229 phase in the 30%-DOPG/ 70%-MO-LUVs at 25 °C, respectively. This finding is the first demonstration of transformation of LUVs of lipid membranes into the cubic phase under excess water condition.
1. Introduction Lipid membranes in cubic phases have nonbilayer structures with connections in three-dimensional (3-D) space, and their membrane structures have a cubic symmetry. Regular three-dimensional structures of biomembranes similar to the cubic phases have been observed in various cells by electron microscopy.1,2 The cubic phase has been postulated to play several important biological roles such as membrane fusion, control of functions of membrane proteins, and ultrastructural organization inside cells. Thereby, elucidation of the mechanism for the stability of cubic phases of biomembranes is recently considered important in the biophysics of biomembrane dynamics and also in the biotechnology of crystallization of proteins and biosensors.1-4 Especially, understanding mechanisms of the phase transitions between cubic phases and LR phase and also finding effective factors to control these phase transitions are very important. Recently, we have systematically investigated the effects of electrostatic interactions due to surface charges in the membrane interface on the structure and the stability of cubic-phase membranes, and have found that, in lipid membranes, the electrostatic interactions due to the surface charges induce transitions between the cubic phase and the LR phase, and also between the cubic phases with different infinite periodic minimal surface (IPMS).5-7 For * To whom correspondence should be addressed. Tel and Fax: 81-54-238-4741. E-mail:
[email protected]. † Graduate School of Science and Engineering, Shizuoka University. ‡ Department of Physics, Faculty of Science, Shizuoka University. (1) Hyde, S.; Andersson, S.; Larsson, K.; Blum, Z.; Landh, T.; Ninham, B. W. The language of shape; Elsevier Science: New York, 1997. (2) Luzzati, V. Curr. Opin. Struct. Biol. 1997, 7, 661. (3) Seddon, J. M.; Templer, R. H. In Structure and dynamics of membranes; Lipowsky, R., Sackmann, E., Eds.; Elsevier Science: Amsterdam, 1995; p 97. (4) Pebay-Peyroula, E.; Rummel, G.; Rosenbusch, J. P.; Landau, E. M. Science 1997, 277, 1676. (5) Aota-Nakano, Y.; Li, S. J.; Yamazaki, M. Biochim. Biophys. Acta 1999, 1461, 96
example, we investigated the structures of DOPA/MO membranes (composed of a mixture of electrically neutral monoolein (MO) and negatively charged dioleoylphosphatidic acid [DOPA]) and found that, as the electrostatic interactions increase (i.e., the surface charge density increases or the salt concentration in the bulk solution decreases), the most stable phase of the MO membrane changes: Q224 f Q229 f LR.6 Later, other groups reported similar results that the MO membranes containing high concentrations of a negatively charged lipid such as dioleoylphosphatidylserine (DOPS) or dioleoylphosphatidylglycerol (DOPG) were in the LR phase.8,9 These results suggested that it is possible to transform small-size vesicles such as large unilamellar vesicles (LUVs) into the cubic phases by the addition of salts. However, in the case of multilamellar vesicles (MLVs) of the DOPA/MO membrane, very high concentrations of NaCl (0.5-0.75M) are required to induce a phase transition from the LR to the Q224 phase. It has been mentioned that Ca2+ can decrease electrostatic interactions in water more effectively than Na+.10 Therefore, it is reasonable to consider that lower concentrations of Ca2+ can induce the LR to the Q224 phase transition in the DOPA/MO membrane. However, Ca2+ did not induce this phase transition. In this report, we investigated the possibility of ioninduced transformation of LUVs of lipid membrane into the cubic phase membranes and have found a good condition. We used the negatively charged DOPG instead of DOPA to give negative charges to the MO membrane interface. At first, we investigated the effect of DOPG on the phase stability of MO membranes by small-angle X-ray scattering (SAXS). We found that the most stable phase changed by increasing the DOPG concentration as fol(6) Li, S. J.; Yamashita, Y.; Yamazaki, M. Biophys. J. 2001, 81, 983. (7) Masum, S. M.; Li, S. J.; Tamba, Y.; Yamashita, Y.; Tanaka, T.; Yamazaki, M. Langmuir 2003, 19, 4745 (8) Cherezov, V.; Clogston, J.; Misquitta, Y.; Abdel-Gawad, W.; Caffrey, M. Biophys. J. 2002, 83, 3393 (9) Chupin, V.; Killian, J. A.; de Kruijff, B. Biophys. J. 2003, 84, 2373 (10) Israerachvili, J. Intermolecular & Surface Forces, 2nd ed.; Academic Press: New York, 1992.
10.1021/la051782i CCC: $30.25 © 2005 American Chemical Society Published on Web 11/02/2005
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lows: Q224 w Q229 w LR. The addition of low concentrations of Ca2+ to the DOPG/MO-MLV induced phase transitions from LR to Q229 or Q,224 depending on Ca2+ concentration. More importantly, we found that low concentrations of Ca2+ transformed the DOPG/MO-LUVs into cubic phases under the same conditions. 2. Materials and Methods 2.1. Materials and Sample Preparation. Monoolein (MO) (1-monooleoyl-rac-glycerol) and tetradecane were purchased from Sigma Chemical Co. (St. Louis, MO). 1,2-Dioleoyl-sn-glycero-3[phospho-rac-(1-glycerol)] (DOPG) sodium salt was purchased from Avanti Polar Lipids. Ethylenediamine-N,N,N′,N′-tetraacetic acid, tetrasodium salt (EDTA) was purchase from Dojindo Laboratories (Kumamoto, Japan). They were used without further purification. Lipid membranes were prepared by adding appropriate amounts of 10 mM PIPES buffer (pH 7.0) to the dry lipids (i.e., mixture of MO and DOPG) in excess PIPES buffer (50 mM lipid concentration) condition or at 30 wt % lipid concentration. These suspensions were then vortexed for about 30 s at room temperature (∼25 °C) several times. For SAXS measurements, pellets after centrifugation (13 000g, 30 min at 20 °C; Tomy, MR-150) of the lipid suspensions were used. To investigate the interaction of Ca2+ with DOPG/MO-MLVs or DOPG/MO membranes in the cubic phase, various concentrations of Ca2+ in 10 mM PIPES buffer (pH 7.0) were added to suspensions of the preformed membranes in the same buffer to bring the total volume of the solution to 200 µL (i.e., 50 mM lipid concentration). The suspensions were then mixed thoroughly by a vortex mixer, and incubated at 25 °C using a temperature-controlled incubator for 72 h ()3 d). After incubation, the suspensions were centrifuged for 15-30 min and the excess buffer was removed by suction. Pellets of the lipids were collected inside a thin glass capillary for the SAXS measurements. To investigate the change in spontaneous curvature of the DOPG/MO monolayer, the structures of DOPG/MO membranes containing 16 wt % tetradecane were measured by SAXS, as described in our previous papers.6,7 To investigate the reversibility of the Ca2+-induced LR to cubic phase transition, after 30%-DOPG/ 70%-MO-MLV changed into the Q224 phase by the 48 h incubation in the presence of 50 mM Ca2+ using the same method described above, the suspension was diluted 10 times with 50 mM EDTA in PIPES buffer. The suspensions were then mixed thoroughly by a vortex mixer and incubated at 25 °C using a temperaturecontrolled incubator for 1-24 h. Pellets of this suspension prepared by the same method described above were used for the SAXS measurement. 2.2. SAXS Measurements. X-ray diffraction experiments were performed by using nickel filtered Cu KR X-ray (λ ) 0.154 nm) from a rotating anode type X-ray generator (Rigaku, Rotaflex, RU-300) at the operating condition (40 kV × 200 mA).5-7 In all cases, samples were sealed in a thin-walled glass capillary tube (outer diameter 1.0 mm) and mounted in a temperature-controlled holder.6,7 The reciprocal spacing, S, of the cubic phase is connected to the lattice constant, a, by the equation S (h, k, l) ) (1/a)‚(h2 + k2 + l2)1/2, where h, k, and l are Miller indices. The lattice constant, a, was determined from the slope of the plot of S vs (h2 + k2 + l2)1/2. 2.3. Interaction of Ions with LUVs of DOPG/MO Membranes. DOPG/MO-LUVs were prepared in 10 mM PIPES buffer (pH 7.0) by the extrusion method using 100 nm pore-size membranes. Samples of 10 mM DOPG/MO-MLV were subjected to five cycles of freeze-thawing in liquid N2, after which the resultant solution was extruded through a 100 nm pore-size membrane using the LiposoFast apparatus (LF-1, Avestin, Ottawa, Canada) until the solution became transparent. Lipid concentrations of the LUV suspensions were determined by the standard Bartlett method. Various concentrations of CaCl2 solutions (300 µL) prepared in 10 mM PIPES (pH 7.0) were mixed thoroughly with 700 µL of the LUV suspension. After incubation of the mixtures at 25 °C, the suspensions were centrifuged for 15 min, and the resulting pellets were collected for the SAXS measurement.
Figure 1. (A) Structure parameter of DOPG/MO membranes containing various concentrations of DOPG (mol %) in 10 mM PIPES buffer (pH 7.0) in excess PIPES buffer at 20 °C determined by SAXS. (0), (4), and (1) denote the Q224, the Q229, and the LR phase, respectively. (B) The basis vector length, d, of HII phases of the DOPG/MO membranes containing 16 wt % tetradecane vs the DOPG concentration (mol %) in 10 mM PIPES buffer (pH 7.0) in excess PIPES buffer at 20 °C determined by SAXS. (0) in the absence of NaCl, and (2) in the presence of 1.0 M NaCl.
3. Results and Discussion 3.1. Effect of DOPG Concentration on Structures of DOPG/MO Membranes. We investigated the effects of DOPG concentration (mol %) in DOPG/MO membranes on their structures in 10 mM excess PIPES buffer (pH 7.0) at 20 °C by SAXS. Figure 1A shows the dependence of the lattice constant and kind of phases on DOPG concentration. At e0.55% DOPG, the DOPG/MO membranes were in the primitive cubic phase of space group Pn3m (i.e., the Q224 phase), whereas at and above 0.60% DOPG, they were in the body-centered cubic phase of space group Im3m (i.e., the Q229 phase). Therefore, a phase transition from the Q224 to the Q229 occurred at 0.60% DOPG. The lattice constant of the Q224 phase immediately before the phase transition (0.55% DOPG) and that of the Q229 phase immediately after the phase transition (0.60% DOPG) were 10.5 and 13.8 nm, respectively. This gives a lattice constant ratio (Q229/Q224) of 1.31, which is close to the theoretical value (1.28) determined by the analysis of the coexisting cubic phases based on the Bonnet transformation.6,11 The lattice constant of the Q229 phasemembrane gradually increased from 13.8 to 18.3 nm with increasing the DOPG concentration from 0.60 to 14%. At 16-23% DOPG, it was difficult to identify the phase from the broad SAXS peaks. At and above 25% DOPG, a new set of SAXS peaks appeared with a large spacing (15.6 ( 0.2 nm) in the ratio of 1:2:3, which is consistent with the LR phase. Therefore, the above results indicated that as the surface charge density of the membrane increased (11) Hyde, S. T.; Andersson, S.; Ericsson, B.; Larsson, K. Z. Kristallogr. 1984, 168, 213
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Figure 2. SAXS patterns of the 30%-DOPG/70%-MO membranes in 10 mM PIPES buffer (pH 7.0) containing various concentrations of Ca2+ at 20 °C. (A) 0 mM Ca2+ at 30 wt % lipid concentration, (B) 25 mM Ca2+ in excess PIPES buffer, (C) 50 mM Ca2+ in excess PIPES buffer. (D) Indexing of SAXS data (panels B and C). (4) 25 mM Ca2+ (B), and (b) 50 mM Ca2+ (C).
(i.e., as the DOPG concentration increases), the most stable phase changed as follows: Q224 w Q229 w LR. This is consistent with the NMR data of Chupin et al. which showed that the DOPG/MO membranes containing high concentrations of DOPG were in the LR phase.9 In contrast, in the presence of 1.0 M NaCl, no phase transitions occurred at 0-30% DOPG; that is, all of the membranes were in the Q224 phase, indicating that electrostatic interactions due to the surface charge induced these phase transitions. These results are almost the same as those of DOPA/MO membranes.6 To consider the mechanism of the phase transitions above-mentioned, we investigated the effects of DOPG concentration on the spontaneous curvature of MO monolayer membrane using the method described in our previous papers.6,12 The basis vector length, d, of the DOPG/MO monolayer membrane containing 16 wt % tetradecane in 10 mM PIPES buffer (pH 7.0) at 20 °C, which is in the HII phase, increased rapidly from 7.0 to 15.6 nm with increasing the DOPG concentration from 0 to 30% (0 in Figure 1B), respectively. It is reasonable to consider that this increase is not only due to the electrostatic repulsive interaction between the negative charges of the membrane but also due to the increase in the content of DOPG in the membrane. To separate the two effects, we investigated the effect of DOPG concentration on the spontaneous curvature of the MO membrane in 10 mM PIPES buffer (pH 7.0) containing 1.0 M NaCl, which decreased the electrostatic interactions in water greatly. As shown in Figure 1B, a slight increase in the d value from 7.0 to 8.0 nm was observed with increasing the DOPG concentration from 0 to 30% (2 in Figure 1B). This slight increase is thus due to the increase in the content of DOPG, which has a small packing parameter (12) Kinoshita, K.; Li, S. J.; Yamazaki, M. Eur. Biophys. J. 2001, 30, 207.
compared with that of MO in the membrane. Accordingly, the increase in d of the DOPG/MO/tetradecane membranes in 10 mM PIPES buffer (0 in Figure 1B) is mainly due to the electrostatic repulsive interaction between the negative charges of the membrane. The increase in d of the DOPG/MO/tetradecane membrane induced by the increase in content of DOPG is attributed to the increase in R0 ) 1/|H0|, since the change in the membrane thickness of the monolayer membrane is considered to be small.6,12 The spontaneous curvature is determined by the packing parameter of lipids, V/Al, where V is the volume of the entire lipid molecule, A is the area of the lipid headgroup at the lipid-water interface, and l is its length.7 The electrostatic repulsive interaction between charged headgroups increases the effective area A, and thereby, this is one of main factors inducing the decrease in |H0| of DOPG/MO monolayer with increasing DOPG concentration. On the basis of our previous papers,6,7 the decrease in |H0| of DOPG/MO monolayer with increasing the electrostatic interactions and the resultant change in the curvature elastic energy of the membrane is one of the main factors to induce the cubic-to-LR phase transition. 3.2. Effect of Ca2+ Concentration on the Phase Stability of DOPG/MO-MLV in the Lr Phase and that of DOPG/MO Membranes in the Q229 Phase. We investigated the effects of Ca2+ concentration in the bulk on the structure and phase of the preformed 30%-DOPG/ 70%-MO-MLV in excess PIPES buffer. In the absence of Ca2+, the 30%-DOPG/70%-MO membrane was in the LR phase in excess (Figure 1A) as well as in low PIPES buffer contents (Figure 2A). Figure 2A shows a SAXS pattern of this MLV at 30 wt % lipid concentration, indicating the LR phase with a spacing of 11.3 nm. In the presence of 25 mM Ca2+, the SAXS pattern revealed several peaks that had spacings in the ratio of x2:x4:x6:x8:x10:x12:x14: x16:x18:x20:x22 (Figure 2B). They were indexed as
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Figure 4. Partial phase diagram of the DOPG/MO membranes, the Ca2+ concentration in solution (mM) versus the DOPG concentration in the membranes (mol %). B denotes the Q224 phase, 4 denotes the Q229 phase, and 1 denotes the LR phase. 0 denotes the coexistence of the Q224 and the Q229 phases.
Figure 3. (A) Structural parameter of the 30%-DOPG/70%MO membranes in 10 mM PIPES buffer (pH 7.0) containing various concentrations of Ca2+ (mM) at 20 °C determined by SAXS. (9) and (4) denote the Q224 and the Q229 phase, respectively. (B) The effect of the Ca2+ concentration on the basis vector length, d, of the HII phases of the 30%-DOPG/ 70%-MO membrane containing 16 wt % tetradecane in excess PIPES buffer at 20 °C determined by SAXS.
(110), (200), (211), (220), (310), (222), (321), (400), (411), (420), and (332) reflections on the Q229 phase. The reflections of (310), (400), and (420) were very small. The reciprocal spacing, S, of the cubic phase is connected to the lattice constant, a, by the equation S (h, k, l) ) (1/ a)‚(h2 + k2 + l2)1/2, where h, k, and l are Miller indices. The lattice constant, a (here called the structure parameter, so that it can be used for different kinds of phases), of the 30%-DOPG/70%-MO membrane in the presence of 25 mM Ca2+ was 15.9 nm (Figure 2D (4)). On the other hand, in the presence of 50 mM Ca2+, several peaks had spacings in the ratio of x2:x3:x4:x6:x8:x9:x10:x11:x12, indexed as (110), (111), (200), (211), (220), (221), (310), (311), and (222) reflections correspond to the Q224 phase (Figure 2C). The reflections of (310) and (311) were very low. The lattice constant was 10.6 nm (Figure 2D (b)). Figure 3A shows a detailed dependence of the lattice constant and phase on the Ca2+ concentration. At
