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Langmuir 2003, 19, 4745-4753

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Effect of de Novo Designed Peptides Interacting with the Lipid-Membrane Interface on the Stability of the Cubic Phases of the Monoolein Membrane Shah Md. Masum,†,‡ Shu Jie Li,†,‡ Yukihiro Tamba,† Yuko Yamashita,§ Tomoki Tanaka,† 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 November 14, 2002. In Final Form: February 26, 2003 Elucidation of the mechanism of transitions between the cubic phase and the liquid-crystalline (LR) phase and between different cubic phases is essential for an understanding of the dynamics of biomembranes and the development of new crystallization techniques. Recently, we found that electrostatic interactions due to the surface charges of lipid membranes induce transitions between the cubic phase and LR phase and between different cubic phases (Aota-Nakano, Y.; Li, S. J.; Yamazaki, M. Biochim. Biophys. Acta 1999, 1461, 96; Li, S. J.; Yamashita, Y.; Yamazaki, M. Biophys. J. 2001, 81, 983). In the present study, we used small-angle X-ray scattering to investigate the effects of a de novo designed peptide (WLFLLKKK, peptide1, which has positive charges and a site that is partitioned in electrically neutral lipid-membrane interfaces) on the stability of the cubic phase of the monoolein (MO) membrane. As the peptide-1 concentration increased, a transition from the Q224 to the Q229 phase in the MO membrane at a 30 wt % lipid concentration occurred at R ) 0.0090 (R is the molar ratio of peptide to MO), and at R g 0.040, the MO/peptide-1 membranes were in the LR phase. As the NaCl concentration in the bulk phase increased, for the MO/ peptide-1 membranes in the Q229 phase in excess water, a Q229 phase to Q224 phase transition occurred at low concentrations of NaCl. Similarly, for the MO/peptide-1 membranes (R ) 0.080) in excess water, at low NaCl concentrations, they were in the LR phase, but at g0.40 M NaCl, they were in the Q224 phase. These results indicate that peptide-1 was partitioned in the membrane interface of the MO membrane, electrostatic interactions due to peptide-1 in the membrane interface make the Q229 phase more stable than the Q224 phase, and with larger electrostatic interactions, the LR phase is more stable than these cubic phases. Increased peptide-1 concentration reduced the absolute value of spontaneous curvature of the MO/peptide-1 monolayer membrane. On the basis of these results, we discuss the mechanism of the effect of peptide-1 on the phase stability of the MO membranes.

1. Introduction The biological and physicochemical aspects of the cubic phases of lipid membranes have attracted much attention. One family of cubic phases, which includes the Q224 phase (Schwartz’s D surface), Q229 phase (P surface), and Q230 phase (G surface), has an infinite periodic minimal surface (IPMS) consisting of bicontinuous regions of water and hydrocarbons.1 In these cubic-phase membranes, the minimal surface (defined to have a zero mean curvature and negative Gaussian curvature at all points) is located at the bilayer midplane (interface between two monolayer membranes). Transmission electron microscopy has revealed regular three-dimensional structures of biomembranes similar to the cubic phases in various cells.1,2 It has been postulated that the cubic phases play important roles in biomembrane dynamics such as membrane fusion, control of membrane protein functions, and various intracellular structures of membranes, such as endoplasmic reticulum.1-5 The most well-known examples of cubic phases are plant-cell prolamellar bodies (PLB), which are considered Q224 or Q229 phase.1,6 After adsorbing light, * Correspondence to Dr. Masahito Yamazaki. Telephone and Fax: +81-54-238-4741. E-mail: [email protected]. † Materials Science, Shizuoka University. ‡ These authors contributed equally. § Department of Physics, Shizuoka University. (1) Hyde, S.; Andersson, S.; Larsson, K.; Blum, Z.; Landh, T.; Ninham, B. W. The Language of Shape; Elsevier Science B.V.: The Netherlands, 1997. (2) Luzzati, V. Curr. Opin. Struct. Biol. 1997, 7, 661.

PLBs transform into thylakoid lamellae, which are in the liquid-crystalline (LR) phase. This transformation is considered a transition from the cubic phase to the LR phase. Recent elegant experiments have shown that cubic phases are very useful for the crystallization of membrane proteins.7,8 Elucidation of the mechanisms of the transitions between the cubic phase and the LR phase and between different cubic phases is essential for an understanding of biomembrane dynamics and the development of new crystallization techniques. However, there has been only limited research on the phase transitions and stability of the cubic phases. The effects of water content and temperature on the stability of cubic phases have been investigated.9-14 Recently, we have systematically investigated the effects of electrostatic interactions due to surface charges on the structure and stability of cubic(3) Colotto, A.; Martin, I.; Ruysschaert, J.-M.; Sen, A.; Hui, S. W.; Epand, R. P. Biochemistry 1996, 35, 980. Colotto, A.; Epand, R. P. Biochemistry 1997, 36, 7644. (4) Basa´n˜ez, G.; Nieva, J. L.; Rivas, E.; Alonso, A.; Gon˜i, F. M. Biophys. J. 1996, 70, 2299. (5) de Kruijff, B. Nature 1997, 386, 129. (6) Li, S. J.; Yamashita, Y.; Yamazaki, M. Biophys. J. 2001, 81, 983. (7) Landau, E. M.; Rosenbusch, J. P. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 14532. (8) Pebay-Peyroula, E.; Rummel, G.; Rosenbusch, J. P.; Landau, E. M. Science 1997, 277, 1676. (9) Anderson, D. M.; Gruner, S. M.; Leibler, S. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 5364. (10) Gruner, S. M. J. Phys. Chem. 1989, 93, 7562. (11) Turner, D. C.; Wanf, Z.-G.; Gruner, S. M.; Mannock, D. A.; McElhaney, R. M. J. Phys. II 1992, 2, 2039. (12) Chung, H.; Caffrey, M. Nature 1994, 368, 224.

10.1021/la026847l CCC: $25.00 © 2003 American Chemical Society Published on Web 04/29/2003

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phase membranes and have found that, in lipid membranes, electrostatic interactions due to surface charges induce transitions between the cubic phase and the LR phase and between different IPMS cubic phases.6,15 However, the mechanisms of phase transition and phase stability and the determination of the structure of biomembrane cubic phases are not well-understood. In cells, proteins and peptides may alter the stability of biomembrane cubic phases.1,2 It has been reported that several peptides, including the so-called fusion peptides of virus membrane proteins, induce the formation of nonbilayer membranes.3,16,17 However, the mechanisms of the effects of peptides and proteins on the stability of cubic phases are not well-understood.18 Recent biophysical studies indicate that the lipid-membrane interface is composed of hydrophilic segments (so-called headgroups), hydrophobic acyl chains, and water molecules incorporated as a result of the large thermal motions of membranes, such as undulation and protrusion.19,20 The thickness of the membrane interface of a dioleoylphosphatidylcholine membrane is ∼1.5 nm, indicating that an R helix can be partitioned in the membrane interface parallel to the bilayer plane.21,22 Factors other than the electrostatic attraction between peptides and membranes are involved in the partitioning of peptides and proteins in the membrane interface of electrically neutral lipid membranes. Free energies of transfer of short peptides from the membrane interface of palmitoyloleoylphosphatidylcholine to water have been obtained experimentally; from the resulting data, an interfacial hydrophobicity scale of amino acid residues (i.e., free energies of transfer of amino acid residues from the membrane interface to water, ∆Gtr) has been constructed.23 These data show that aromatic amino acid residues such as tryptophan (W) and phenylalanine (F) have a high interfacial hydrophobicity (∆Gtr of tryptophan is 1.85 kcal/mol and ∆Gtr of phenylalanine is 1.13 kcal/mol), indicating a strong partitioning of these residues in the lipid-membrane interface. In a previous study, we designed and synthesized a peptide with the sequence WLFLLKKK (peptide-1). Peptide-1 has positive charges and can be partitioned in a lipid-membrane interface composed of electrically neutral lipids (Figure 1). We investigated the effects of peptide-1 on phosphatidylcholine (PC) membranes.24 The N-terminal region (WLFLL) of peptide-1 is partitioned in the membrane interface because F and W have a high interfacial hydrophobicity, even though this peptide has four positive charges at a neutral pH. The free energy of transfer of (13) Seddon, J. M.; Templer, R. H. In Structure and dynamics of membranes; Lipowsky, R., Sackmann, E., Eds.; Elsevier Science B.V.: Amsterdam, The Netherlands, 1995; p 97. (14) Templer, R. H.; Seddon, J. M.; Warrender, A.; Syrykh, A.; Huang, Z.; Winter, R.; Erbes, J. J. Phys. Chem. B 1998, 102, 7251. Templer, R. H.; Seddon, J. M.; Duesing, P. M.; Winter, R.; Erbes, J. J. Phys. Chem. B 1998, 102, 7262. (15) Aota-Nakano, Y.; Li, S. J.; Yamazaki, M. Biochim. Biophys. Acta 1999, 1461, 96. (16) Keller, S. L.; Gruner, S. M.; Gawrisch, K. Biochim. Biophys. Acta 1996, 1278, 241. (17) Siegel, D. P.; Epand, R. M. Biochim. Biophys. Acta 2000, 1468, 87. (18) Epand, R. M. Lipid Polymorphism and Membrane Properties. In Current Topics in Membranes; Epand, R. M., Ed.; Academic Press: San Diego, 1998; p 237. (19) Israerachvili, J. Intermolecular & Surface Forces, 2nd ed.; Academic Press: New York, 1992. (20) Kinoshita, K.; Furuike, S.; Yamazaki, M. Biophys. Chem. 1998, 74, 237. (21) Wiener, M. C.; White, S. H. Biophys. J. 1992, 61, 434. (22) White, S. H.; Wimley, W. C. Curr. Opin. Struct. Biol. 1994, 4, 79. (23) Wimley,v W. C.; White, S. H. Nat. Struct. Biol. 1996, 3, 842. (24) Yamashita, Y.; Masum, S. M.; Tanaka, T.; Yamazaki, M. Langmuir 2002, 18, 9638.

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Figure 1. Schematic view of the membrane interface of the MO membrane and the partition of the peptide-1 into the membrane interface. The lipid membrane consists of two membrane interfaces and the hydrophobic core (see details in the text). Peptide-1 is located in the membrane interface.

peptide-1 from the membrane interface to water has been estimated to be 1.69 kcal/mol (using the following ∆Gtr values: leucine (L) +0.56 kcal/mol and lysine (K) - 0.99 kcal/mol; we neglected the effects of peptide termini; i.e., NH3+ and CONH2), indicating that peptide-1 can be partitioned in the membrane interface of PC bilayers. In contrast, the free energy of transfer of peptide-2 (LLKKK, used for control experiments) from the membrane interface to water was estimated to be -1.85 kcal/mol, indicating that peptide-2 is not partitioned in the membrane interface of PC bilayers. Peptide-1 was designed to bring positive charges to the membrane interface of electrically neutral lipid membranes as a result of its partition. Peptide-1 increased the intermembrane distance of the PC multilamellar vesicles (MLV) and induced various shape changes in giant PC liposomes, whereas peptide-2 did not have any effects on PC membranes, indicating that peptide-1 was appropriately designed.24 In the present study, we investigated the effects of peptide-1 on the stability of the cubic phases of the monoolein (MO) membranes. MO membranes are in the Q224 phase in excess water over a wide range of temperatures.25 In previous studies, we investigated the structures of dioleoylphosphatidic acid (DOPA)/MO membranes (composed of a mixture of MO and negatively charged DOPA)6 and oleic acid (OA)/MO membranes (composed of a mixture of MO and negatively charged OA)15 to elucidate the effects of electrostatic interactions resulting from the surface charge of membranes on the stability of the cubic phases. As the electrostatic interactions increase (i.e., the surface charge density increases or salt concentration in the bulk phase decreases), the most stable phase of a MO membrane changes: Q224 f Q229 f LR. Subsequently, we have begun to investigate the effects of peptides (or proteins) on the stability of the cubic phases. First, we investigated the effects of peptides interacting with the membrane interface of a MO membrane. Little information is available regarding the structure of the membrane interface of a MO membrane. However, a recent molecular dynamics simulation shows that there is a great deal of undulation motion and peristaltic motion in MO membranes.26 This suggests that the membrane interface of a MO membrane is composed of hydrophilic segments, hydrophobic acyl chains, and water molecules, which is similar to the composition of a PC membrane. Therefore, it is to be expected that a peptide designed to be partitioned in the membrane interface of a PC membrane is applicable to MO membranes. To verify this hypothesis, in the present study, we investigated the effects of peptide-1 on the structures and stability of the cubic phase of MO membranes, using small-angle X-ray scattering (SAXS). We expected that peptide-1 would be partitioned in the membrane interface of a MO membrane and bring positive charges to the membrane interface (Figure 1), which would change the stability of the cubic phase of the MO (25) Qiu, H.; Caffrey, M. Biomaterials 2000, 21, 223. (26) Marrink, S. J.; Mark, A. E. J. Phys. Chem. B 2001, 105, 6122.

Monoolein Membrane

membrane as a result of electrostatic interactions. We found that, as the peptide-1 concentration increased, a transition from the Q224 to the Q229 phase occurred in the MO/peptide-1 membrane. At higher peptide-1 concentrations, the MO/peptide-1 membranes were in the LR phase. On the basis of these findings, we discuss the mechanisms of the effects of peptide-1 on the stability of the cubic phase of MO membranes. 2. Materials and Methods Materials and Sample Preparation. MO (1-monooleoylrac-glycerol) and tetradecane were purchased from Sigma Chemical Co. (St. Louis, MO). Trifluoroethanol (TFE) was purchased from Wako Chemical Co. Piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES) was purchased from Dojindo Molecular Technologies, Inc. These chemicals were used without further purification. The lipid membranes were prepared as follows. MO dissolved in chloroform and various concentrations of peptides dissolved in TFE were mixed, dried using N2, and then kept under vacuum by a rotary vacuum pump for more than 12 h. An appropriate amount of a 10 mM PIPES buffer (pH 7.0) containing a given concentration of NaCl was added to the resulting dry powder mixture of MO and peptide, at an ∼7 wt % lipid concentration (high excess of water) or a 30 wt % lipid concentration. Then, these lipid suspensions were vortexed for about 30 s at room temperature (∼25 °C) several times, followed by incubation at room temperature for 4 h (for the lipid suspension in the absence of NaCl) and 12 h (for the lipid suspensions containing NaCl solutions). For the measurement of X-ray diffraction, the lipid suspensions prepared at a high excess of water were centrifuged (13 000 × g, 30 min, 20 °C; Tomy MR-150), and the resulting pellets were used as samples in excess water because the supernatant above the pellet maintained the excess-water condition. The lipid suspensions prepared at 30 wt % lipids were not centrifuged before being used for X-ray diffraction. To investigate the structures of a MO membrane/peptide-1 mixture containing 16 wt % tetradecane, we used a method similar to those described in previous papers.6,27,28 All procedures were performed at room temperature. An appropriate amount of MO containing various concentrations of peptide-1 in chloroform/TFE was dried using N2 and was then dried under vacuum by a rotary pump for more than 12 h. Tetradecane was added to the dry lipid by direct weighing, followed by vortexing for about 30 s several times. After 48 h of incubation for equilibration, an appropriate amount of a 10 mM PIPES buffer (pH 7.0) containing a given concentration of NaCl was added to this drylipid/peptide/tetradecane mixture in excess water, and this suspension was vortexed for about 30 s several times. Then, it was incubated for another 24 h for equilibration. For the measurement of X-ray diffraction, we precipitated the suspensions after vortexing without centrifugation; that is, the samples for X-ray diffraction were under excess-water conditions. Peptide Synthesis and Purification. The peptides were synthesized by the FastMoc method using a 433A peptide synthesizer (PE Applied Biosystems, Foster City, CA). The sequence of peptide-1 (8-mer) is WLFLLKKK, and that of peptide-2 is LLKKK. These peptides have an amide-blocked C terminus. The methods of purification and identification of peptides were the same as those we used in previous studies.24,29 Peptides were purified by reversed-phase high-performance liquid chromatography (LC-10AD and SPD-10A, Shimazu, Kyoto, Japan) using a C18 semipreparative column (10 × 250 mm, 10 µm) and a C18 analytical column (4.6 × 250 mm, 5 µm; Vydac/ The Separation Group Inc., Hesperia, CA). The purified peptides were analyzed by ion-spray ionization mass spectrometry using a single quadrupole mass spectrometer (API 150EX, PE SCIEX, PE Applied Biosystems, Foster City, CA); ionization was performed at a flow rate of 5 µL/min. The measured masses of peptide-1 and peptide-2 were 1074.2 ( 0.3 and 627.8 ( 0.1 Da, (27) Kinoshita, K.; Li, S. J.; Yamazaki, M. Eur. Biophys. J. 2001, 30, 207. (28) Chen, Z.; Rand, R. P. Biophys. J. 1998, 74, 944. (29) Oblatt-Montal, M.; Yamazaki, M.; Nelson, R.; Montal, M. Protein Sci. 1995, 4, 1490.

Langmuir, Vol. 19, No. 11, 2003 4747 respectively. These masses correspond to the molecular masses calculated from their amino acid compositions. X-ray Diffraction. X-ray diffraction experiments were performed using nickel-filtered Cu KR X-rays (λ ) 0.154 nm) from a rotating anode-type X-ray generator (Rigaku, Rotaflex, RU300) at 40 kV and 200 mA. SAXS data were recorded using a linear (one-dimensional) position-sensitive proportional counter (Rigaku, PSPC-5)30 with a camera length of 350 mm and associated electronics (multichannel analyzer, etc.; Rigaku). In all cases, samples were sealed in a thin-walled glass capillary tube (outer diameter 1.0 mm) and mounted in a thermostatable holder with a stability of (0.2 °C. 27,31 Formation and Observation of the Giant Unilamellar Vesicle (GUV). GUVs of the MO/peptide-1 membrane were prepared using the standard method for the preparation of GUVs6,24,32 as follows. A mixture of MO and peptide-1 in chloroform/TFE was dried in a small glass vessel, using a stream of nitrogen gas at room temperature. Then, the residual solvent was completely removed by a rotary vacuum pump for more than 12 h. A small amount of water (about 10 µL) was added to the glass vessel, followed by incubation at 45 °C for a few minutes (prehydration). Then, 100 µL of 0.1 M sucrose in a 10 mM PIPES buffer (pH 7.0) was added to the vessel, followed by incubation at 37 °C for 2 h. A 40-µL sample of the GUV solution was diluted in 300 µL of 0.1 M glucose in a 10 mM PIPES buffer (pH 7.0), and this solution was placed in a handmade microchamber. The method of observation of the GUVs using a Hoffmann modulation contrast microscope is described in a previous paper.6

3. Results Effects of the Peptide-1 Concentration on the Structure and Phase of the MO/Peptide-1 Membranes. We investigated effect of peptide-1 (WLFLLKKK) on the structure of the MO membrane in a 10 mM PIPES buffer (pH 7.0) at 30 wt % lipid (i.e., 70 wt % water) at 20 °C by SAXS. At >50 wt % water, the MO membrane is in the Q224 phase over a wide temperature range. The addition of small amounts of peptide-1 to the MO membrane changed this cubic structure. The SAXS pattern of the MO/peptide-1 membrane (R ) 0.0060, where R is molar ratio of peptide to MO) was similar to that of 100% MO membrane (Figure 2A). Several peaks had spacing with a ratio of x2:x3:x4:x6:x8:x9:x10:x14, indexed as (110), (111), (200), (211), (220), (221), (310), and (321) reflections (Figure 3). This corresponds to a primitive cubic phase of space group Pn3m (Q224 phase) or Pn3 (Cubic aspects #4; International Tables for X-ray Crystallography, 1985). 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 MO/peptide-1 membrane (R ) 0.0060) was 10.8 nm (Figure 3). In contrast, in the SAXS pattern of the MO/peptide-1 membrane (R ) 0.010) (Figure 2B), several peaks had spacing with a ratio of x2:x4:x6:x12:x14:x16:x18:x22: x24:x26. They were indexed as (110), (200), (211), (222), (321), (400), (411), (332), (422), and (431) reflections on a body-centered cubic phase of space group Im3m (Q229 phase; Cubic aspects #8). The lattice constant of this membrane was 14.7 nm (Figure 3). As was mentioned by Tenchov et al.,33 it is difficult to distinguish Im3m from Pn3n (Q222; Cubic aspects #6) using only the SAXS pattern because the only difference in the SAXS patterns between these structures is the presence of the x21 reflection (30) Glatter, O.; Kratky, O. Small-Angle X-ray Scattering; Academic Press: New York, 1982. (31) Yamazaki, M.; Ohshika, M.; Kashiwagi, N.; Asano, T. Biophys. Chem. 1992, 43, 29. (32) Tanaka, T.; Tamba, Y.; Masum, S. M.; Yamashita, Y.; Yamazaki, M. Biochim. Biophys. Acta 2002, 1564, 173.

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Figure 4. Structure parameter of MO/peptide-1 membranes in the presence of various concentrations of peptide-1 (b, 2, 1) and peptide-2 (O) in a 10 mM PIPES buffer (pH 7.0) at 30 wt % lipid concentration at 20 °C determined by SAXS. R is the molar ratio of peptides to MO. b and O denote the Q224 phase, 2 denotes the Q229 phase, and 1 denotes the LR phase. Table 1. Structure, Phase, and Spontaneous Curvature of MO/Peptide-1 Membranes in a 10 mM PIPES Buffer (pH 7.0; 30 wt % Lipid) peptide concentration R

Figure 2. SAXS patterns of MO/peptide-1 membranes in a 10 mM PIPES buffer (pH 7.0) at 30 wt % lipid concentration at 20 °C. (A) R ) 0.0060, (B) R ) 0.010, and (C) R ) 0.10. R is the molar ratio of peptide-1 to MO. Only the strong peaks were labeled with indices in the figure.

a

Figure 3. Indexing of SAXS data (Figure 2) of MO/peptide-1 membranes in a 10 mM PIPES buffer (pH 7.0) at 30 wt % lipid concentration at 20 °C. (A) R ) 0.0060; (b) strong intensity peak, (O) weak intensity peak. (B) R ) 0.010; (9) strong intensity peak, (0) weak intensity peak.

in Pn3n and because soft materials in water such as lipid membranes do not have high S/N of SAXS peaks in the relatively high-angle region around the x21 reflection. However, the lattice constant ratio (Q229/Q224) at the peptide-induced transition from the Q224 to the Q229 phase is in good agreement with the theoretical value estimated for the transformation between these IPMSs (see next paragraph). This supports our identification of the phase as Im3m. Figure 4 and Table 1 show the dependence of the lattice constant and cubic-phase type of the MO/peptide-1 membrane at 30 wt % lipid on the concentration of peptide1. At R < 0.0080, MO membranes were in the Q224 phase. At R g 0.0090, MO membranes were in the Q229 phase. Thus, at R ) 0.0090, a transition from the Q224 to the Q229

phase

structure parameter a (nm)

+ tetradecane dcc (nm) of HII phasea

0 V 0.0080

Q224

10.2 ∧ 11.2

6.7 ∧ ∧

0.0090 0.010 0.020 0.030

Q229

14.5 14.8 17.4 18.7

6.9 7.4 ∧

0.040 0.060 V 0.12

LR

10.8 9.2 ∨ 8.2

7.8 7.9 ∧ 8.5

See Figure 8A and its description in the text for details.

phase occurred. The lattice constant of the Q224 phase immediately before the phase transition (R ) 0.0080) and that of the Q229 phase immediately after the phase transition (R ) 0.0090) were 11.2 and 14.5 nm, respectively. The lattice constant ratio (Q229/Q224) was 1.29, which is close to the theoretical value (1.28) determined by the analysis of coexisting cubic phases based on the Bonnet transformation among IPMSs.33,34 The lattice constant of the MO/peptide-1 membrane in the Q229 phase gradually increased from 14.5 to 18.7 nm as the peptide-1 concentration increased from R ) 0.0090 to 0.030. At R g 0.040, a new set of SAXS peaks appeared with large spacing (1:2:3 ratio; Figure 2C), which is consistent with a lamellar LR phase. The spacing (i.e., structure parameter) of the LR phase decreased slightly as the peptide-1 concentration increased from R ) 0.040 to 0.12 (Figure 4). In the LR phase, there was no excess of water because the intermembrane distance of the MO/peptide-1 MLV was very large because the electrostatic repulsion between the membrane was large and the water content was not sufficiently high (70 wt % water). Moreover, peptide-1 interacts with water favorably. Thereby, the spacing did not increase with an increase in peptide-1 concentration. As a control experiment, the effect of peptide-2 (LLKKK) on the structure of the MO membranes has been investigated at a 30 wt % lipid concentration. In the C-terminal region, peptide-2 has the same sequence as peptide-1, but peptide-2 does not have the highly interfacially hydro(33) Tenchov, B.; Koynova, R.; Rapp, G. Biophys. J. 1998, 75, 853. (34) Hyde, S. T.; Andersson, S.; Ericsson, B.; Larsson, K. Z. Kristallogr. 1984, 168, 213.

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Figure 5. Hoffmann modulation contrast images of the GUVs of the MO/peptide-1 membrane (R ) 0.10), which were prepared in a 10 mM PIPES buffer (pH 7.0). The scale bar is 20 µm.

phobic site, WLF, at its N terminus. Therefore, it is reasonable to expect that peptide-2 is not partitioned in the membrane interface. The SAXS data show that the structure and phase (Q224) of the MO membrane was not changed by the presence of peptide-2 at R < 0.12 (Figure 4). The effects of peptide-1 on the structure and phase of the MO/peptide-1 membranes in a 10 mM PIPES buffer (pH 7.0) in excess water was already reported.35 At R < 0.011, the membranes were in the Q224 phase, and at 0.011 e R e 0.040, the membranes were in Q229 phase.35 The slight difference in results between 30 wt % lipid and excess water suggests that the water content affects the stability of these cubic phases. At R g 0.060, the SAXS pattern peaks became broad and, therefore, difficult to analyze. This phenomenon is similar to that observed in DOPA/MO membranes; in excess water, the LR phase was not specified as a result of a broad SAXS peak, but it was specified at 30 wt % lipid.6 To confirm the formation of the LR phase in the MO/peptide-1 membrane containing a high concentration of peptide-1 at a high excess of water, we attempted to construct a GUV using this membrane and successfully constructed GUVs at around R ) 0.10. Most of these GUVs were spherical vesicles 20-50 µm in diameter (Figure 5). These results indicate that the MO/ peptide-1 membranes containing a high concentration of peptide-1 at a high excess of water were in the LR phase and could, therefore, form GUVs. In contrast, MO membranes containing peptide-2 could not form GUVs. These findings are consistent with the results of SAXS. Effects of the Salt Concentration on the Lattice Constants and Cubic-Phase Types in MO/Peptide-1 Membranes. To elucidate the effects of the electrostatic interactions on the phase stability of the MO/peptide-1 membrane, we investigated dependence of the NaCl concentration in the bulk phase on the structure of the MO/peptide-1 membrane in excess water. Figure 6A shows details of the dependence of the lattice constant and phase types of the MO/peptide-1 membrane (R ) 0.030) on the NaCl concentration. At e110 mM NaCl, the membranes were in the Q229 phase. In contrast, at g120 mM NaCl, they were in the Q224 phase. Thus, at 120 mM NaCl, a phase transition from Q229 to Q224 occurred. The lattice constant of the Q229 phase immediately before the phase transition (110 mM NaCl) and that of the Q224 phase immediately after the phase transition (120 mM NaCl) were 15.6 and 12.2 nm, respectively. The lattice constant ratio (Q229/Q224) was 1.28, which is equal to the theoretical value.33,34 The lattice constant of the Q224 phase gradually decreased as the NaCl concentration increased. A similar

result was obtained for MO/peptide-1 membranes (R ) 0.020) in excess water: at 20 mM NaCl, they were in the Q224 phase.35 Next, we investigated the dependence of the NaCl concentration on the structures of the MO/peptide-1 membrane (R ) 0.080) in excess water. As was mentioned previously, in 0 M NaCl, it was in the LR phase (Figure 4). At 0.1-0.3 M NaCl, a set of SAXS peaks appeared with large spacing (1:2 ratio), which is consistent with an LR phase. The spacing (i.e., structure parameter) of the LR phase decreased slightly (from 10.4 to 9.9 nm) as the NaCl concentration increased from 0.1 to 0.3 M. The SAXS pattern showed that, at g0.4 M NaCl, the membranes were in the Q224 phase and the lattice constant of the Q224 phase gradually decreased as the NaCl concentration increased (Figure 6B). Effects of Peptide-1 on the Spontaneous Curvature of MO Monolayer Membranes. To consider the mechanism of the phase transitions previously mentioned, we investigated effects of the peptide-1 concentration on the spontaneous curvature of the MO monolayer membrane. The spontaneous curvature of a single monolayer membrane, H0, is a useful parameter characterizing nonbilayer membranes and is expressed as H0 ) 1/R0, where R0 is the radius of spontaneous curvature.27,36,37 We have a useful method to get information on the

(35) Li, S. J.; Masum, S. M.; Yamashita, Y.; Tamba, Y.; Yamazaki, M. J. Biol. Phys. 2002, 28, 253.

(36) Gruner, S. M. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 3665. (37) Marsh, D. Biophys. J. 1996, 70, 2248.

Figure 6. (A) Lattice constant of the cubic phases of the MO/ peptide-1 membranes (R ) 0.030) in a 10 mM PIPES buffer (pH 7.0) containing various concentrations of NaCl (M) in excess water at 20 °C determined by SAXS. 4 denotes the Q229 phase and O denotes the Q224 phase. (B) Structure parameter of the MO/peptide-1 membrane (R ) 0.080) in a 10 mM PIPES buffer (pH 7.0) containing various concentrations of NaCl (M) at 20 °C determined by SAXS. 3 denotes the LR phase and O denotes the Q224 phase.

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Figure 7. SAXS pattern of MO/peptide-1 membranes containing 16 wt % tetradecane in a 10 mM PIPES buffer (pH 7.0) in excess water at 20 °C. (A) R ) 0; pure MO membrane without peptide-1. (B) R ) 0.060.

spontaneous curvature of the lipid membrane. To allow the lipid monolayer membranes in the hexagonal II (HII) phase (or inverted hexagonal phase) in excess water to express the spontaneous curvature, the addition of alkanes such as decane and tetradecane to the membranes is required because they fill the interstitial region of the HII phase and relax the hydrocarbon-chain packing stress.36,38 Under this condition, the curvature of the monolayer membrane in the HII phase is very close to the spontaneous curvature. As in a previous study,6 MO membranes containing g8 wt % tetradecane in excess water at 20 °C in a 10 mM PIPES buffer (pH 7.0) were in the HII phase (Figure 7A), and the basis vector length (distance between the centers of adjacent cylinders in the HII phase), dcc, of these MO/tetradecane membranes (6.7 nm) changed very little as the tetradecane concentration increased from 8 to 24 wt %. To get information of the dependence of the spontaneous curvature of the MO membrane on the peptide-1 concentration, we investigated the structure of the MO/peptide-1 membrane containing 16 wt % tetradecane (i.e., MO/peptide-1/tetradecane membrane) in excess water at 20 °C. In excess water at 20 °C, dcc of the MO/ peptide-1/tetradecane membrane in a 10 mM PIPES buffer (pH 7.0) gradually increased from 6.7 to 9.0 nm as the peptide-1 concentration increased from R ) 0 to 0.15 (Figures 7B and 8A). Generally, dcc of the HII phase changes as the tetradecane concentration increases because the additional tetradecane fills the interstitial void. Above a critical tetradecane concentration, the basis vector length no longer increases. It is at these tetradecane concentrations that the measurement of the spontaneous curvature of the monolayer membrane should be performed. Below the critical tetradecane concentration, the MO membrane is in the cubic phase; after the transition from the cubic to the HII phase, dcc does not change appreciably between 8 and 24 wt % tetradecane. This indicates that our experiments using 16 wt % tetradecane should yield accurate information on the spontaneous curvature of the monolayer MO membrane. We also investigated the effect of the NaCl concentration on the structure of the MO/peptide-1 membrane (R ) 0.080) containing 16 wt % tetradecane in a 10 mM PIPES buffer (pH 7.0) in excess water (Figure 8B). In 0 M NaCl, the MO/peptide-1 membrane (R ) 0.080) without tetradecane was in the LR phase, but the MO/peptide-1 membrane (R ) 0.080) containing 16 wt % tetradecane was in the HII phase (dcc ) 8.2 nm). As is shown in Figure 8B, dcc gradually decreased from 8.2 to 6.7 nm as the NaCl concentration increased from 0 to 1.0 M. (38) Rand, R. P.; Fuller, N. L.; Gruner, S. M.; Parsegian, V. A. Biochemistry 1990, 29, 76.

Figure 8. (A) The basis vector length, dcc, of HII phases of MO membranes containing 16 wt % tetradecane versus peptide-1 concentration (R) in a 10 mM PIPES buffer (pH 7.0) in excess water at 20 °C determined by SAXS. (B) Effect of the NaCl concentration on dcc of HII phases of MO/peptide-1 membranes (R ) 0.080) containing 16 wt % tetradecane in a 10 mM PIPES buffer (pH 7.0) in excess water at 20 °C determined by SAXS. Table 2. Effect of the NaCl Concentration on the Structure, Phase, and Spontaneous Curvature of MO/ Peptide-1 Membranes (R ) 0.080) in a 10 mM PIPES Buffer (pH 7.0) in Excess Water NaCl concentration

a

phase

0 0.10 0.20 0.30

LR

0.40 0.50 0.60

Q224

structure parameter a (nm)

+ tetradecane dcc (nm) of HII phasea

10.4 10.4 9.9

8.2 ∨ 7.7 ∨

13.5 12.4 11.7

7.4 ∨ 7.1

See Figure 8B and its description in the text for details.

4. Discussion Effects of Peptide-1 on the Phase Stability and Structure of the MO Membrane, and Partition of Peptide-1 in its Membrane Interface. The present X-ray diffraction results clearly show that peptide-1 induced changes in the phase and structure of the MO membrane; at low critical concentrations of peptide-1, a Q224 phase to Q229 phase transition occurred; at higher concentrations of peptide-1, the LR phase becomes more stable than the Q229 phase in the MO membranes. Moreover, high concentrations of NaCl inhibit these phase changes (Table 2 and Figure 9). These results are similar to those of our previous studies.6,15 In those studies, to elucidate the effects of the electrostatic interactions resulting from the membrane surface charge (e.g., DOPA and OA) on the structure and stability of the cubic phases of the MO membrane, we investigated the structure of DOPA/MO membranes and OA/MO membranes. As the electrostatic interactions increase (i.e., surface charge density increases or salt concentration in the bulk phase

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Figure 9. Partial phase diagram of MO/peptide-1 membranes, the NaCl concentration (M) in solution versus peptide-1 concentration 〈R〉. O denotes the Q224 phase, 4 denotes the Q229 phase, and 1 denotes the LR phase.

decreases), the most stable phase of the MO membrane changes: Q224 f Q229 f LR. Therefore, we can reasonably interpret the present results as follows. The partition of peptide-1 in the membrane interface of the MO membrane occurs at the N-terminal region (WLFLL) of peptide-1 as a result of its high interfacial hydrophobicity (Figure 1). Peptide-1 increases the surface charge density of the MO membrane because peptide-1 has three positive charges at its C terminus and one positive charge at its N terminus. As the peptide-1 concentration increases (and surface charge density of the membrane consequently increases), the electrostatic repulsion between the positive charges increases. In contrast, in the MO/peptide-1 membrane, as the salt concentration in the bulk phase increases, the electrostatic repulsion decreases as a result of the screening of the charges of the peptides. Therefore, the present results clearly show the following. The Q229 phase is more stable than the Q224 phase in the MO membrane in excess water when there is a strong electrostatic repulsion between the positive charges of the peptides partitioned in the membrane interface. When the peptide-1 concentration increases (and the electrostatic interactions consequently increase), the LR phase becomes more stable than the Q229 phase (Figure 10). What effects of these electrostatic interactions are important in the phase stability of the MO/peptide-1 membrane? Effects of Peptide-1 on the Spontaneous Curvature of MO Monolayer Membranes. The spontaneous curvature of a single monolayer membrane is defined as the curvature that minimizes its curvature elastic energy, which is determined by the physical properties of the monolayer without interaction with other monolayer membranes.27,36,37 Therefore, it is a kind of ideal curvature of the monolayer membrane. In most cases, it is difficult to construct a lipid membrane with spontaneous curvature because interaction between two monolayer membranes also plays an important role in the determination of the curvature. The main determinant of the spontaneous curvature of a single monolayer membrane is the geometric packing of the constituent lipids.19,37 The spontaneous curvature of the HII phase membrane is characterized by a packing parameter, 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, as follows:37

|H0| ) 1/R0 )

(1 - c)/l cV/Al - 1 - x(cV/Al)2 - 2cV/Al + c

(1)

Figure 10. Effect of the electrostatic interactions on the phase stability of the cubic phases and LR phase. The surface charge density of the lipid membrane can be controlled by the concentration of the charged lipids in the membrane or by the amount of the charged peptides partitioned in the membrane interface. As the electrostatic interactions increase (i.e., the surface charge density of the membrane increases or the salt concentration in the bulk phase decreases), the most stable phase of the MO membrane changes: Q224 f Q229 f LR. This phase change is deeply correlated with the change of the spontaneous curvature of the monolayer lipid membrane. As |H0| of the membrane decreases, the transition from the Q224 to the Q229 phase occurs, and then the Q229-to-LR-phase transition occurs (Table 1).

where c is constant: π/(2x3) < c < 2π/(3x3). |H0| decreases as V/Al decreases.37 Values of these parameters (V, A, l) depend not only on the molecular structure of the lipids but also on the external conditions such as the temperature and solvents (including salts) because these external conditions can greatly change the optimal values of these parameters (V, A, l).27,37 Therefore, the spontaneous curvature of the monolayer membrane depends on both the molecular structure of the constituent lipids and the various external conditions, as was verified in previous studies.27,37 The basis vector length of the HII phase, dcc, is expressed as the sum of the radius to the neutral plane, Rpp, and the distance between the bilayer midplane and the neutral plane, ξ: dcc ) 2(Rpp + ξ).27,38 The neutral plane is the appropriate surface for definition of the curvature of the membrane because the area of this plane remains constant as the monolayer is bent. In excess water, the curvature of the MO/peptide-1/tetradecane membrane is very close to the spontaneous curvature, H0; thus, Rpp ≈ R0. The large increase in dcc of the MO/peptide-1/tetradecane membrane with increasing peptide-1 concentration is

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attributable to the increase in Rpp because the change in ξ is assumed to be small. Thus, the results shown in Figure 8A indicate that the increase in the electrostatic repulsion between peptides reduces the absolute value of spontaneous curvature, |H0|, of the membrane; this increases the distance between the positive charges of different peptides partitioned in the membrane interface. These results are consistent with eq 1, which predicts that |H0| decreases as the packing parameter decreases as a result of the increase in A. Thus, the effect of peptide-1 on the spontaneous curvature of the MO membrane is the same as that of DOPA.6 The present analysis revealed a correlation between the spontaneous curvature of the monolayer membrane and the phase stability of the MO membrane. As |H0| of the MO monolayer membrane decreases, the transition from the Q224 to the Q229 phase occurs and then the transition from the Q229 to the LR phase occurs (Table 1). Stability of the Cubic Phases. Differences in the chemical potential of the phospholipid membrane between the nonbilayer phases [such as the cubic phases (Q224, Q229) and the HII phase; µnonbil] and the LR phase (µbil), ∆µ, are expressed as follows:6,9,36

∆µ ) µnonbil - µbil bil nonbil ) (µnonbil - µbil curv - µcurv) + (µch ch )

(2)

) ∆µcurv + ∆µch where ∆µcurv is a term resulting from the curvature elastic energy (or curvature energy) and ∆µch is a term resulting from interstitial chain packing of the nonbilayer phase. The bilayer midplane of the bicontinuous cubic phase membranes (such as the Q224 and Q229 phases), in which direction the ends of the lipid acyl chains face, is considered to be the IPMS. Thus, each point on their midplane has a zero mean curvature (H ) 0) and a negative Gaussian curvature (K < 0). From the Gauss-Bonnet theorem, the average value of the Gaussian curvature over the area of the unit cell, 〈K〉, is expressed as 〈K〉 ) 2πχ/S, where χ is the Euler number (χ ) 2 - 2g; g is genus of the surface) and S is the area of the membrane surface of the unit cell.39,40 To factor in the curvature elastic energy of the lipid membranes, we have to calculate the change in curvature at the neutral plane where the cross-sectional area of the lipid does not change during the changes in curvature.11,12 The curvature elastic energy of the membrane, µcurv, can be expressed as follows:12,36

µcurv ) 2κ〈H - H0〉2 + κG〈K〉

(3)

where κ is the elastic bending modulus, H is the mean curvature, K is the Gaussian curvature, κG is the Gaussian curvature modulus, and 〈 〉 is the average value over the area of the unit cell. The cubic-phase membranes, such as those in the Q224 and Q229 phases, have a zero mean curvature at all points in the bilayer midplane, but the neutral plane of the membrane can be considered to have a constant nonzero mean curvature because it is at a fixed distance from the minimal surface.9,10 Because H ) K ) 0 in the LR phase, we obtain the following:

∆µcurv ) 2κ(〈H - H0〉2 - H02) + κG〈K〉

(4)

(39) Kobayashi, S. Differential Geometry of Curves and Surfaces; Shokabo: Tokyo, 1995. (40) Hyde, S. T. J. Phys. Chem. B 1989, 93, 1458.

Although κG has a negative value,41 〈K〉 < 0, and, thereby, the second term of eq 4 is positive. However, the absolute value of the negative first term of eq 4 is much larger than that of the second term in most cases because H0 < H < 0 for these nonbilayer membranes. Thus, eq 4 shows that ∆µcurv is negative (∆µcurv < 0) in most cases and, thereby, is an important factor in stabilizing the nonbilayer phases. In contrast, in the HII phase, the hydrocarbon chains of the lipids have to extend to different lengths to fill the interstitial hydrocarbon region, thus reducing the entropy of the chains; consequently, the free energy of the membrane increases.9 This situation is almost the same in the cubic phases. Therefore, the packing energy of these hydrocarbon chains unstabilizes the nonbilayer phases, and, thus, ∆µch is always positive (∆µch > 0). In most cases, the transitions between the nonbilayer phases and the LR phases are determined by the interplay between ∆µcurv and ∆µch. Equation 4 shows that |∆µcurv| decreases as |H0| decreases. For the MO membrane, |H0| is large, and, consequently, ∆µcurv has a large negative value. Therefore, ∆µ < 0, indicating that the cubic phase is stable. As the electrostatic repulsion increases as a result of peptide-1 partitioned in the membrane interface, |H0| of the membrane decreases, inducing a decrease in |∆µcurv|. At the critical value of the electrostatic interactions, ∆µ ) 0, and, consequently, the cubic-to-LR-phase transition occurs. Above the critical value of the electrostatic interactions, ∆µ > 0, and, consequently, the LR phase is stable. Thus, the decrease in |H0| that accompanies an increase in the electrostatic interaction between peptides in the membrane interface is a main factor in the cubic-to-LR-phase transition in the MO/peptide-1 membranes. Development of this theory of the mechanisms of the electrostaticinteraction-induced cubic-to-LR-phase transition will require further data, such as contributions of the changes in the Gaussian curvature of the membranes and quantitative estimation of factors such as the electrostatic interaction between the membranes. For the peptide-1-induced transition from the Q224 to the Q229 phase, the present results indicate that, as the electrostatic interactions increase, the Q229 phase becomes more stable than the Q224 phase and that, as |H0| of the membrane decreases, the transition from the Q224 to the Q229 phase occurs. There have been several proposed theories and quantitative analyses for the effects of the water content in unit cells of cubic phases on the stability of the IPMS cubic phases [Q224 phase (D surface), Q229 phase (P surface), and Q230 phase (G surface)].11,12,14 Those studies have shown that, as the water content increases, the most stable phase changes in the sequence Q230 f Q224 f Q229 (i.e., G f D f P) as a result of changes in the curvature elastic energy. In the present study, as the electrostatic interactions due to the surface charge of membranes increased, the water content in the unit cells of the cubic phases may have increased, and, consequently, the most stable phase changes in the sequence Q224 f Q229 (i.e., D f P) as a result of changes in the curvature elastic energy. Elucidation of the mechanism of the transition between the Q224 and the Q229 phases will require experimental data such as the elastic bending modulus κ, Gaussian curvature modulus κG, and effects of the electrostatic interactions due to the surface charge on the values of κ and κG. It will also require theories for the quantitative estimation of other factors such as the electrostatic interaction between membranes. (41) Templer, R. H.; Khoo, B. J.; Seddon, J. M. Langmuir 1998, 14, 7427.

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The effects of the water content and temperature on the stability of the cubic phases are well-understood.1,13,25 However, other determinants of the stability and structure of the cubic phases are not well-understood. As was described previously, we recently proposed that the electrostatic interactions resulting from the surface charges of lipid membranes play an important role in the stability and structure of the cubic phases of lipid membranes and also in the transition between the cubic phase and the LR phase.6,15 Other recent studies have found that the stability of the cubic phase of lipid membranes can be controlled via the mixing of cationic and anionic lipids;42,43 this supports the hypothesis that the electrostatic interaction in the lipid-membrane interface is a determinant of the stability of the cubic phases. In this study, we have clearly shown that peptide-1 partitioned in the membrane interface can change the stability of the cubic phases and induce the cubic-to-LR-phase transition as a result of the electrostatic repulsion between the positive charges of the peptides partitioned in the membrane interface. Thus, the present findings also indicate that the electrostatic interaction in the lipid-membrane interface is a determinant of the stability of the cubic phases (Figure 10). Biological Implications. Cubic phases are thought to play important roles in the biological membranes of cells.1-3 It has been postulated that, in cells, cubic-to-LRphase transitions occur in several situations, such as the transformation of a PLB.1,6 The mechanism of the cubicto-LR-phase transition in cells is not well-understood. We propose the following hypothesis for the mechanism of (42) Lewis, R. N. A. H.; McElhaney, R. N. Biophys. J. 2000, 79, 1455. (43) Tarahovsky, Y. S.; Arsenault, A. L.; MacDonald, R. C.; McIntosh, T. J.; Epand, R. M. Biophys. J. 2000, 79, 3193.

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the cubic-to-LR-phase transition. In cells, the incorporation of anionic lipids into biomembranes or partition of peptides or proteins with a large net charge on the lipid-membrane interface increase the surface charge density of the biomembranes; consequently, the electrostatic interactions increase. At a critical level of electrostatic interaction, the cubic-to-LR-phase transition occurs (Figure 10). We also propose a hypothesis regarding the importance of the cubic-to-LR-phase transition in cells. Using cubic phases, cells can store a large area of lipid membrane at a high density as a result of the three-dimensional-network structure of the cubic phase. When cells need a large area of the bilayer lipid membrane, a cubic-to-LR-phase transition is induced, instantly producing a large area of the LR-phase membrane. This process occurs much faster than the biogenesis of biomembranes by synthesizing new lipids. We believe that this hypothesis offers a reasonable explanation of the biogenesis of biomembranes such as the thylakoid membrane from PLB. Besides the cubic phases, in cells, various curvatures of biomembranes and changes in the curvature play important roles in the formation of various biomembrane structures and dynamic structural change, such as membrane fusion and budding. In the present study, we have clearly shown that peptide-1 partitioned in the membrane interface can change the spontaneous curvature of the membranes. Cells may contain proteins and peptides that play similar roles. Further study on the effect of peptides and proteins on them is necessary. Acknowledgment. This work was supported in part by a Grant for Basic Science Research Projects from the Sumitomo Foundation (Japan) to M.Y. LA026847L