Effect of Positively Charged Short Peptides on Stability of Cubic

836 Oya, Shizuoka 422-8529, Japan, and Department of Physics, Faculty of Science,. Shizuoka University, Shizuoka, 422-8529, Japan. Received December 1...
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Effect of Positively Charged Short Peptides on Stability of Cubic Phases of Monoolein/Dioleoylphosphatidic Acid Mixtures Shah Md. Masum,† Shu Jie Li,† Tarek S. Awad,† 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 December 10, 2004. In Final Form: March 15, 2005 To elucidate the stability and phase transition of cubic phases of biomembranes with infinite periodic minimal surface is indispensable from biological and physicochemical aspects. In this report, we investigated the effect of positively charged peptide-3K (LLKKK) and poly(L-lysine) on the phase stability of monoolein (MO) membranes containing negatively charged dioleoylphosphatidic acid (DOPA) (i.e., DOPA/MO membranes) using small-angle X-ray scattering. At first, the effect of peptide-3K on 10% DOPA/90% MO membrane in excess water, which is in the Q229 phase, was investigated. At 3.4 mM peptide-3K, a Q229 to Q230 phase transition occurred, and at >3.4 mM peptide-3K, the membrane was in the Q230 phase. Poly(L-lysine) (Mw 1K-4K) also induced the Q230 phase, but peptide-2K (LLKK) could not induce it in the same membrane. We also investigated the effect of peptide-3K on the multilamellar vesicle (MLV) of 25% DOPA/75% MO membrane, which is in LR phase. In the absence of peptide, the spacing of MLV was very large (11.3 nm), but at g8 mM peptide-3K, it greatly decreased to a constant value (5.2 nm), irrespective of the peptide concentration, indicating that peptide-3K and the membranes form an electrostatically stabilized aggregation with low water content. Poly(L-lysine) also decreased greatly the spacing of the 25% DOPA/75% MO MLV, indicating the formation of a similar aggregation. To compare the effects of peptide3K and poly(L-lysine) with that of osmotic stress on stability of the cubic phase, we investigated the effect of poly(ethylene glycol) with molecular weight 7500 (PEG-6K) on the phase stability of 10% DOPA/90% MO membrane. With an increase in PEG-6K concentration, i.e., with an increase in osmotic stress, the most stable phase changed as follows; Q229 (Schwartz’s P surface) w Q224 (D) w Q230 (G). On the basis of these results, we discuss the mechanism of the effects of the positively charged short peptides (peptide-3K) and poly(L-lysine) on the structure and phase stability of DOPA/MO membranes.

1. Introduction Cubic phases of biomembranes have attracted much attention in both biological and physicochemical aspects.1-6 Three-dimensional (3-D) regular structures of biomembranes similar to cubic phases have been observed in various cells by transmission electron microscopy.4,6 They have been postulated to play several important biological roles such as membrane fusions, a control of functions of membrane proteins, and ultrastructural organizations inside cells.3,4,7 One family of cubic phases, which includes 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 hydrocarbon.2 In these cubic phase membranes, the minimal surface (defined to have zero mean curvature and negative Gaussian curvature at all points) is located * Correspondence should be addressed to Dr. Masahito Yamazaki, Department of Physics, Faculty of Science, Shizuoka University, 836 Oya, Shizuoka 422-8529, Japan. TEL and FAX: 81-54-2384741. E-mail: [email protected]. † Materials Science, Graduate School of Science and Engineering, Shizuoka University. ‡ Department of Physics, Faculty of Science, Shizuoka University. (1) Lindblom, G.; L. Rilfors. Biochim. Biophys. Acta 1989, 988, 221. (2) Seddon, J. M.; Templer, R. H. In Structure and dynamics of membranes; Lipowsky, R., Sackmann, E., Eds.; Elsevier Science B.V.: Amsterdam, 1995; pp 97-160. (3) Luzzati, V. Curr. Opin. Struct. Biol. 1997, 7, 661-668. (4) Hyde, S.; Andersson, S.; Larsson, K.; Blum, Z.; Landh, T.; Ninham, B. W. The language of shape; Elsevier Science B.V.: Amsterdam, 1997. (5) Pebay-Peyroula, E.; Rummel, G.; Rosenbusch, J. P.; Landau, E. M. Science 1997, 277, 1676. (6) Li, S. J.; Yamashita, Y.; Yamazaki, M. Biophys. J. 2001, 81, 983. (7) de Kruijff, B. Nature 1997, 386, 129.

at the bilayer midplane (interface between two monolayer membranes). To elucidate the physiological roles of the cubic phase of biomembranes and the mechanism of the above phenomena, an understanding of the phase stability of the cubic phase membranes is necessary; however, little is known.2,3,8 Several factors controlling stability of cubic phase membranes have been reported. Among them, temperature and water content have been vigorously investigated and several temperature-water concentration phase diagrams of lipids were determined.2,9,10 Recently, we investigated the phase stability of monoolein (MO) membranes containing negatively charged lipid (i.e., anionic lipid), such as membranes of dioleoylphosphatidic acid/MO mixtures (DOPA/MO membranes) and those of oleic acid/MO mixtures (OA/MO membranes), and on the basis of this research we have shown that electrostatic interactions due to surface charge of the membrane play an important role in phase stability of cubic phases and also phase transition between cubic phase and lamellar liquid-crystalline (LR) phase.6,11 As the electrostatic interactions in the membrane interface are increased either by the increase in surface charge density of the membrane or by the decrease in salt concentration, the most stable phase of these lipid membranes changes as follows: Q224 w Q229 w LR. Later, other groups reported similar results (8) Anderson, D. M.; Gruner, S. M.; Leibler, S. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 5364. (9) Templer, R. H.; Seddon, J. M.; Warrender, N. A.; Syrykh, A.; Huang, Z.; Winter, R.; Erbes, J. J. Phys. Chem. B 1998, 102, 7251. (10) Qiu, H.; Caffrey, M. Biomaterials 2000, 21, 223. (11) Aota-Nakano, Y.; S. J. Li, S. J.; Yamazaki, M. Biochim. Biophys. Acta 1999, 1461, 96.

10.1021/la0469607 CCC: $30.25 © 2005 American Chemical Society Published on Web 05/04/2005

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on the effects of negatively charged lipids (DOPS, cardiolipin, DOPG) on the phase stability of the MO membrane.12,13 More recently, a different approach for the investigation of the effects of electrostatic interactions on the stability of cubic phase has been done. To increase the surface charge density of the MO membrane, a charged peptide which can be bound with the electrically neutral membrane interface14 was used instead of the negatively charged lipids.15,16 As the peptide concentration increased and, thereby, the surface charge density of the MO membrane due to the bound peptide in the interface increased, the most stable phase of the MO membrane changed as follows: Q224 w Q229 w LR.15,16 In biological cells, proteins and peptides may change stability of the cubic phases of biomembranes. Several peptides such as the so-called fusion peptides of membrane proteins in viruses have been reported to induce nonbilayer membranes such as the cubic phase and hexagonal (HII) phase.17-20 However, the mechanism of the effects of peptides and proteins on the stability of cubic phases is not well understood.21 On the other hand, it is reported that many water-soluble proteins can be bound with lipid membrane regions in biomembranes reversibly and that their binding depends on their concentration in aqueous phase, their conformation, and their local net charge. Especially, the electrostatic interaction between a cluster of basic amino acid residues of proteins and negatively charged lipid membranes and also the hydrophobic interaction between fatty acid covalently linked protein and lipid membranes are well understood, and they play an important role in the binding of proteins with biomembranes.22,23 For example, myristoylated alanine-rich C kinase substrate (MARCKS) and src (pp60src) can be bound with lipid membranes using both the electrostatic attractive interaction and the hydrophobic interaction due to the covalently linked fatty acid. As described above, the investigation of DOPA/MO membranes gave us information of effects of electrostatic interactions on stability of cubic phases. Thereby, a further study on a system of DOPA/MO membrane will give us more insight on the stability of cubic phases of biomembranes. In this report, we investigated two factors to control the phase stability of 10 mol % DOPA/90 mol % MO membrane (i.e., 10% DOPA/90% MO), which is in the Q229 phase at neutral pH in excess water.6 At first, we investigated effects of basic peptides which are bound with negatively charged DOPA/MO membranes in excess water. As the basic peptides, LLKKK (i.e., peptide-3K), LLKK (i.e., peptide-2K), and poly(L-lysine) with molecular (12) Cherezov, V.; Clogston, J.; Misquitta, Y.; Abdel-Gawad, W.; Caffrey, M. Biophys. J. 2002, 83, 3393. (13) Chupin, V.; Killian, J. A.; de Kruijff, B. Biophys. J. 2003, 84, 2373. (14) Yamashita, Y.; Masum, S. M.; Tanaka, T.; Yamazaki, M. Langmuir 2002 18, 9638. (15) Li, S. J.; Masum, S. M.; Yamashita, Y.; Tamba, Y.; Yamazaki, M. J. Biol. Phys. 2002 28, 253 (16) Masum, S. M.; Li, S. J.; Tamba, Y.; Yamashita, Y.; Tanaka, T.; Yamazaki, M. Langmuir 2003 19, 4745. (17) Colotto, A.; Martin, I.; Ruysschaert, J.-M.; Sen, A.; Hui, S. W.; Epand, R. P. Biochemistry 1996, 35, 980. (18) Colotto, A.; Epand, R. P. Biochemistry 1997, 36, 7644. (19) Keller, S. L.; Gruner, S. M.; Gawrisch, K. Biochim. Biophys. Acta, 1996, 1278, 241. (20) Siegel, D. P.; Epand, R. M. Biochim. Biophys. Acta, 2000, 1468, 87. (21) Epand, R. M. Lipid Polymorphism and Membrane Properties. In Current Topics in Membranes; Epand, R. M., Ed.; Academic Press: San Diego, CA, 1998; Vol. 44, pp 237-252. (22) Kim, J.; Blackshear, P. J.; Johnson, D.; McLaughlin, S. Biophys. J. 1994, 67, 227. (23) Buser, C. A.; Sigal, C. T.; Resh, M. D.; McLaughlin, S. Biochemsitry 1994, 33, 13093.

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weight of 1K-4K were used. At their critical concentrations, peptide-3K induced the Q229 to Q230 phase transition and poly(L-lysine) stabilized the Q230 phase. To consider mechanism of these effects, we investigated effects of peptide-3K and poly(L-lysine) on 25% DOPA/75% MO multilanellar vesicle (MLV), which is in the LR phase under the same condition. In the presence of lower concentrations of peptide-3K and poly(L-lysine), spacings of the MLV were very small and constant irrespective of the peptide concentration. Next, we investigated effects of osmotic stress on phase stability of the 10% DOPA/90% MO membranes in excess water. We can control the chemical potential of water inside the membrane by the osmotic stress using poly(ethylene glycol) (PEG).24-26 We found that the most stable phase changed as follows: Q229 (Schwartz’s P surface) w Q224 (D) w Q230 (G) with an increase in PEG-6K concentration, i.e., with an increase in osmotic stress. On the basis of these results, we discuss the mechanism of the effect of the positively charged short peptides (i.e., peptide-3K and poly(L-lysine)) on structure and phase stability of the 10% DOPA/90% MO membrane in the Q229 phase. 2. Materials and Methods 2.1. Materials. Monoolein (MO) (1-monooleoyl-rac-glycerol) and poly(L-lysine) hydrobromide (molecular weight 1000-4000, P0879) were purchased from Sigma Chemical Co. (St. Louis, MO). Dioleoylphosphatidic acid (DOPA) sodium salt was purchased from Avanti Polar Lipids. Poly(ethylene glycol) (PEG) with molecular weight 7500 (PEG-6K) was purchased from Wako Pure Chemical Industry Ltd. They were used without further purification. 2.2. Peptide Synthesis and Purification. Peptides were synthesized by the FastMoc method using a 433A peptide synthesizer (PE Applied Biosystems, Foster City, CA). The sequence of peptide-3K is LLKKK (i.e., +H3N-Leu-Leu-Lys-LysLys-CONH2), and that of peptide-2K is LLKK (i.e., +H3N-LeuLeu-Lys-Lys-CONH2). These peptides have an amide-blocked C terminus. Methods of purification and identification of peptides were the same as those we used in previous studies.14,16 Peptides were purified by reversed-phase high-performance liquid chromatography (LC-10AD & 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-2 and peptide-3 were 627.8 ( 0.1 Da and 499.8 ( 0.1 Da, respectively. These masses correspond to the molecular mass calculated from their amino acid composition. 2.3. Preparation of Membranes. Lipid membranes in aqueous solution were prepared as follows. The appropriate amount of MO in chloroform and that of DOPA in chloroform were mixed. The chloroform solution of the mixture was dried by N2 gas and then under vacuum by rotary pump for more than 12 h. Appropriate amounts of 10 mM PIPES buffer (pH 7.0) were added to the dry lipid thin film in excess water (100 mM lipid concentration) or at 30 wt % lipid concentration. Then, the suspensions were vortexed for about 30 s at room temperature (∼25 °C) several times. For experiments of the interactions of peptide-3K (or peptide2K) with DOPA/MO membranes, 20 mM peptide-3K (or peptide2K) in 10 mM PIPES buffer (pH 7.0) was prepared. Various amounts of peptide-3K (or peptide-2K) solution were added to suspensions containing the preformed DOPA/MO membranes in 10 mM PIPES buffer (pH 7.0) in the excess water (final concentration of lipids was 50 mM) as described above and mixed (24) Ito, T.; Yamazaki, M.; Ohnishi, S. Biophys. J. 1989, 56, 707. (25) Yamazaki, M.; Ohshika, M.; Kashiwagi, N.; Asano, T. Biophys. Chem. 1992, 43, 29. (26) Chung, H.; Caffrey, M. Nature 1994, 368, 224.

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by a vortex mixer. After the mixing, the suspensions were incubated at room temperature (∼25 °C) for 24 h. Then, they were centrifuged at 13000g for 30 min at 20 °C, and the resulting pellets were used as samples in excess water for X-ray diffraction, because the supernatant above the pellet maintained the excess water condition. For experiments of the interactions of poly(L-lysine) with DOPA/MO membranes, 200 or 800 mM poly(L-lysine) were prepared in 10 mM PIPES buffer (pH 7.0). Various amounts of poly(L-lysine) solution were added to suspension containing the preformed DOPA/MO membranes in 10 mM PIPES buffer (pH 7.0) in the excess water (final concentration of lipids was 50 mM) as described above and mixed by a vortex mixer. After the mixing, the suspensions were incubated at room temperature (∼25 °C) for 48 h. Then they were centrifuged at 13000g for 30 min at 20 °C, and the resulting pellets were used as samples in excess water for X-ray diffraction, because the supernatant above the pellet maintained the excess water condition. We also investigated the effect of freeze-thawing of samples on the results of smallangle X-ray scattering (SAXS). After the mixing, these suspensions were frozen in liquid N2 for 2 min and then thawed at room temperature for 30 min. This freeze-thawing was repeated three times to equilibrate the suspensions. The SAXS results of samples prepared using the freeze-thawing were almost the same as those of samples prepared without the freeze-thawing. For experiments of the interactions of PEG-6K with DOPA/ MO membranes, various concentrations of PEG-6K in 10 mM PIPES buffer (pH 7.0) were added to the dry lipids, i.e., mixtures of MO and DOPA, in excess water (100 mM lipid concentration) and mixed by a vortex mixer. After the mixing, the suspensions were incubated at room temperature (∼25 °C) for 1 h, after which they were centrifuged. 2.4. 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, RU-300) at 40 kV and 200 mA. SAXS data were recorded using a linear (one-dimensional) position-sensitive proportional counter (PSPC) (Rigaku, PSPC-5)27 with 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 (1.0 mm outer diameter) and mounted in a thermostatable holder with a stability of (0.2 °C.25

3. Results and Discussion 3.1. Effects of Peptide-3K (LLKKK) on 10% DOPA/ 90% MO Membranes. We investigated effects of peptide3K on 10% DOPA/90% MO membrane in 10 mM PIPES buffer (pH 7.0) in excess water at 20 °C. Under this condition, the membrane was in a body-centered cubic phase of space group Im3m (Q229) (cubic aspect #8).6 We added peptide-3K solution in 10 mM PIPES buffer (pH 7.0) into this preformed membrane in the Q229 phase and measured this structure by SAXS after 24 h of incubation. At low concentrations of peptide-3K, the 10% DOPA/90% MO membranes were also in the Q229 phase. For example, in a SAXS pattern of this membrane at 3.0 mM peptide3K (Figure 1A), several peaks had spacings in the ratio of x2:x4:x6:x8:x10:x12:x14:x16:x18:x20:x22, which were indexed as (110), (200), (211), (220), (310), (222), (321), (400), (411), (420), and (332) reflections, indicating that the membrane was in the Q229 phase (Figure 2A).6 Intensities of the reflections of (400), (420), and (332) were very weak. The reciprocal spacing, S, of the cubic phase is connected with the lattice constant, a, by S (h,k,l) ) (1/a)(h2 + k2 + l2)1/2, where h, k, and l are Miller indices. The lattice constant, a, of this membrane at 3.0 mM peptide-3K was 13.6 nm (Figure 3). In contrast, in the SAXS pattern of 10% DOPA/90% MO membrane in the presence of 8.0 mM peptide-3K, several peaks had spacings in the ratio of x6:x8:x14:x16:x20:x22:x24 (Figure 1C), (27) Glatter, O.; Kratky, O. Small-Angle X-ray Scattering; Academic Press: San Diego, CA, 1982.

Figure 1. X-ray diffraction profiles of 10% DOPA/90% MO membrane in excess water in 10 mM PIPES buffer (pH 7.0) at 20 °C: (A) at 3.0 mM; (B) at 3.4 mM; (C) at 8.0 mM peptide-3K. From the indexing analysis, (A) shows the Q229 phase, (C) shows the Q230 phase, and (B) shows the coexistence of both the phases. In (B), peaks with by asterisks (/) are due to the Q229 phase and peaks with pound signs (#) are due to the Q230 phase.

indexed as (211), (220), (321), (400), (420), (332), and (422) reflections on a body-centered cubic phase of space group Ia3d (Q230) (cubic aspect #12) (Figure 2A). Intensity of the reflection of (420) was very weak. The lattice constant of this Q230 phase was 15.1 nm (Figure 3). Figure 3 shows a detailed dependence of the structure of 10% DOPA/90% MO membranes on peptide-3K concentration, indicating that a phase transition from Q229 to Q230 occurred at 3.4 mM peptide-3K, where the two phases coexisted (Figure 1B and Figure 2B). The lattice constants for the Q229 and Q230 phases were 12.8 and 16.0 nm, respectively (Figure 3). The ratio of their lattice constant (Q230/Q229) was 1.25, which is almost the same as the theoretical value (1.23) determined by the analysis of the coexisting cubic phases based on the Bonnet transformation.28,29 This also supports that the Q229 to Q230 phase transition occurred at 3.4 mM (28) Tenchov, B.; Koynova, R.; Rapp, G. Biophys. J. 1998, 75, 853. (29) Hyde, S. T.; Andersson, S.; Ericsson, B.; Larsson, K. Z. Kristallogr.1984, 168, 213.

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Figure 4. X-ray diffraction profiles of 10% DOPA/90% MO membrane at 22 mM poly(L-lysine) in excess water in 10 mM PIPES buffer (pH 7.0) at 20 °C.

Figure 2. Indexing of SAXS data (Figure 1) of 10% DOPA/ 90% MO membrane in 10 mM PIPES buffer (pH 7.0) in excess water at 20 °C: (A) (9) Figure 1A (at 3.0 mM peptide-3K) and (0) Figure 1C (at 8.0 mM peptide-3K); (B) Figure 1B at 3.4 mM peptide-3K, (0) for / peaks and (9) for # peaks.

Figure 5. The lattice constant of cubic phases of 10% DOPA/ 90% MO membranes in the presence of various concentrations of poly(L-lysine) in 10 mM PIPES buffer (pH 7.0) in excess water at 20 °C determined by SAXS: (0) denotes the Q229 phase; (3) denotes the Q230 phase. Shading by slant lines indicates the nonequilibrium area.

Figure 3. The lattice constant of cubic phases of 10% DOPA/ 90% MO membranes in the presence of various concentrations of peptide-3K in 10 mM PIPES buffer (pH 7.0) in excess water at 20 °C determined by SAXS: (O) denotes the Q229 phase, and (0) denotes the Q230 phase.

peptide-3K. As a control experiment, we investigated effects of peptide-3K on 100% MO membranes in 10 mM PIPES buffer (pH 7.0) in excess water at 20 °C. The phase and its lattice constant did not change up to 10 mM. The mechanism of this Q229 to Q230 phase transition will be discussed later. 3.2. Effects of Poly(L-lysine) and Peptide-2K (LLKK) on 10% DOPA/90% MO Membranes. Next, we investigated effects of poly(L-lysine) (Mw 1000-4000) on 10% DOPA/90% MO membrane in 10 mM PIPES buffer (pH 7.0) in excess water at 20 °C. We added poly(L-lysine) solution in 10 mM PIPES buffer (pH 7.0) into this preformed membrane in the Q229 phase and measured this structure by SAXS after 48 h of incubation. Figure 4 shows a SAXS pattern of 10% DOPA/90% MO membrane in the presence of 22 mM poly(L-lysine); several peaks had spacings in the ratio of x6:x8:x14:x16:x20:x22, which were indexed as (211), (220), (321), (400), (420), and (332) reflections on a body-centered cubic phase of space group Ia3d (Q230). The lattice constant of this Q230 phase was 13.4 nm (Figure 5). Figure 5 shows a detailed

dependence of structure of 10% DOPA/90% MO membranes on poly(L-lysine) concentration. At high concentrations of poly(L-lysine), i.e., at g16 mM, 10% DOPA/90% MO membranes were in the Q230 phase and the lattice constant of the membranes were the same (13.5 ( 0.2 nm) irrespective of poly(L-lysine) concentration up to 200 mM (data above 40 mM not shown). At lower concentrations of poly(L-lysine), we could not specify a phase due to complicated SAXS patterns, probably because it was very difficult to attain equilibrium condition. We also tried freeze-thawing of samples for SAXS measurement to attain equilibrium, but the SAXS patterns of samples prepared using the freeze-thawing were almost the same as those of samples prepared without the freeze-thawing. As a control experiment, we investigated effects of poly(L-lysine) on 100% MO membranes in 10 mM PIPES buffer (pH 7.0) in excess water at 20 °C. The phase and its lattice constant did not change irrespective of poly(L-lysine) concentration up to 200 mM. These results show that the effects of poly(L-lysine) on 10% DOPA/90% MO membrane are the same as that of peptide-3K (LLKKK). It suggests that any peptides containing a segment of KK‚‚‚K ) (K)n (for peptide-3K, n ) 3, and for poly(L-lysine) n ) 8-30) would induce the Q230 phase in 10% DOPA/90% MO membrane. To determine the minimum number of the lysine residues (i.e., the minimum number of positive charges due to the lysine residues), n, we investigated effects of peptide-2K (LLKK) (n ) 2) on 10% DOPA/90% MO membrane in 10 mM PIPES buffer (pH 7.0) in excess water at 20 °C. Figure 6A shows a SAXS pattern of 10% DOPA/90% MO membrane in the presence of 10 mM peptide-2K; several

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Figure 6. (A) X-ray diffraction profiles of 10% DOPA/90% MO membrane in excess water in 10 mM PIPES buffer (pH 7.0) at 20 °C in the presence of 10 mM peptide-2K. (B) The lattice constant of cubic phases of 10% DOPA/90% MO membranes in the presence of various concentrations of peptide-2K in 10 mM PIPES buffer (pH 7.0) in excess water at 20 °C determined by SAXS: (O) denotes the Q229 phase; (0) denotes the Q224 phase.

peaks had spacings in the ratio of x2:x3:x4:x6:x8:x9, indexed as (110), (111), (200), (211), (220), and (221) reflections, which corresponds to the primitive cubic phase of space group Pn3m (Q224) (cubic aspect #4). Figure 6B shows a detailed dependence of structure of 10% DOPA/ 90% MO membranes on peptide-2K concentration. At 8 mM peptide-2K, a Q229 to Q224 phase transition occurred. This type of phase transition was observed in the interaction of NaCl with 10% DOPA/90% MO membrane. Thereby, we can conclude that the minimum number of the lysine residues (n) of the peptide to induce the Q230 phase is 3. 3.3. Effects of Peptide-3K (LLKKK) and Poly(Llysine) on 25% DOPA/75% MO MLV. Next, we investigated effects of peptide-3K on 25% DOPA/75% MO membranes in 10 mM PIPES buffer (pH 7.0) in excess water at 20 °C. A SAXS pattern of this membrane in the absence of peptide-3K under the same condition has a broad peak, which is difficult to assign a phase. However, Figure 7A shows that, at 30 wt % lipid concentration, a set of SAXS peaks appeared with a large spacing (dl ) 11.3 nm) in the ratio of 1:2:3, indicating that the 25% DOPA/75% MO membrane was in the LR phase under this nonexcess water condition. As reported in a previous paper, giant unilamellar vesicles of this membrane were formed in excess water.6 On the basis of these results, we could reasonably conclude that this membrane in excess water is in the LR phase.6 In excess water, presence of low concentrations of peptide-3K changed greatly the SAXS pattern of the 25% DOPA/75% MO membrane. For example, in the presence of 10 mM peptide-3K in excess water, a set of sharp SAXS peaks appeared with a small spacing (dl ) 5.2 nm) in the ratio of 1:2:3 (Figure 7B), indicating that this membrane was in the LR phase. Figure 8A shows a detailed dependence of the spacing of the 25%

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Figure 7. X-ray diffraction profiles of 25% DOPA/75% MO membranes in 10 mM PIPES buffer (pH 7.0) at 20 °C: (A) 30 wt % lipid concentration (in the absence of peptide-3K); (B) in 10 mM peptide-3K in excess water.

Figure 8. Spacing of 25% DOPA/75% MO membranes in the presence of various concentrations of (A) peptide-3K and (B) poly(L-lysine) in 10 mM PIPES buffer (pH 7.0) in excess water at 20 °C determined by SAXS. Shading by slant lines indicates the nonequilibrium area.

DOPA/75% MO membranes on peptide-3K concentration in bulk phase. At g8 mM peptide-3K, a SAXS pattern of the LR phase appeared. The spacings were almost constant

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(5.2 ( 0.1 nm), irrespective of peptide-3K concentration from 8 to 20 mM. At lower concentrations (