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 Q 229 phase, was investigated. At 3.4 mM peptide-3K, a Q 229 to Q 230 phase transition occurred, and at >3.4 mM peptide-3K, the membrane was in the Q 230 phase. Poly(L-lysine) (Mw 1K-4K) also induced the Q 230 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 peptide- 3K 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; Q 229 (Schwartz’s P surface) w Q 224 (D) w Q 230 (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 biomem- branes 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 Q 224 phase (Schwartz’s D surface), Q 229 phase (P surface), and Q 230 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 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, temper- ature 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 (L R ) phase. 6,11 As the electrostatic in- teractions 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: Q 224 w Q 229 w L R . Later, other groups reported similar results * Correspondence should be addressed to Dr. Masahito Yamaza- ki, Department of Physics, Faculty of Science, Shizuoka University, 836 Oya, Shizuoka 422-8529, Japan. TEL and FAX: 81-54-238- 4741. E-mail: spmyama@ipc.shizuoka.ac.jp. † 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. (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. 5290 Langmuir 2005, 21, 5290-5297 10.1021/la0469607 CCC: $30.25 © 2005 American Chemical Society Published on Web 05/04/2005