Langmuir 1999, 15, 4461-4463
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Vesicular Self-Assembly of a Helical Peptide in Water Shunsaku Kimura,*,† Do-Hyung Kim,† Junji Sugiyama,‡ and Yukio Imanishi§ Department of Material Chemistry, Graduate School of Engineering, Kyoto University, Yoshida Honmachi, Sakyo-ku, Kyoto 606-8501, Japan, Institute of Wood Science, Kyoto University, Gokanosho, Uji, Kyoto 611-0011, Japan, and Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara 630-0101, Japan Received December 3, 1998. In Final Form: April 6, 1999 The formation of a molecular assembly composed of a naturally occurring peptide with a helical structure was investigated. Gramicidin A, a 15-mer peptide antibiotic, was conjugated with poly(ethylene glycol) (PEG, average molecular weight 600). This peptide conjugate formed vesicles with an unilamellar membrane in water as shown by frozen-hydrated/cryo-transmission electron microscopy (cryo-TEM) observations. The peptide fragment adopted an antiparallel double-helix conformation. The vesicles of the gramicidin conjugate encapsulating PEG showed much greater stability than encapsulation by dimyristoylphosphatidylcholine liposomes with respect to resistance to collapse upon detergent addition. Since the peptide membrane core is constituted of the secondary structured units, the peptide vesicles (named peptosomes) may be advantageous to functional membranes due to their highly regular structure.
Molecular assemblies have attracted much attention in relation to a wide range of biological phenomena and the development of novel materials.1 Recently, the relationship between the morphology of the molecular assembly and the structure of low-molecular-weight amphiphiles2 is considered to be applicable to the highermolecular-weight amphiphiles.3 Indeed, block copolymers were reported to adopt multiple morphologies in water, such as micelles, fibrils, and vesicles.4,5 For example, amphiphilic block copolymers containing a poly(styrene) tail and a charged helical poly(isocyanide) headgroup selfassembled to form various aggregates including helical superstructures which can be controlled by variation of the length of the poly(isocyanide) block.6 However, in most cases the polymer chains were just random-coil conformations in the core region of the copolymer assemblies. In contrast, we report here a naturally occurring peptide derivative that spontaneously forms self-organized vesicles with a regular helix conformation in the membrane-core region. Gramicidin A (g.A), a 15-mer peptide antibiotic, is known to form ion channels in lipid bilayer membranes with a helical structure.7-12 g.A is composed entirely of hydrophobic amino acids and is therefore partitioned to the †
Department of Material Chemistry, Kyoto University. Institute of Wood Science, Kyoto University. § Nara Institute of Science and Technology. ‡
(1) Fuhrhop, J.-H.; Keoning, J. Membranes and molecular assemblies: The synkinetic approach; Cambridge University Press: Cambridge, UK, 1994. (2) Cullis, P. R.; De Kruijff, B. Biochim. Biophys. Acta 1979, 559, 399-420. (3) Van Hest, J. C. M.; Delnoye, D. A. P.; Baars, M. W. P. L.; van Genderen, M. H. P.; Meijer, E. W. Science 1995, 268, 1592-1595. (4) Yu, K.; Eisenberg, A. Macromolecules 1996, 29, 6359-6361. (5) Zhang, L.; Eisenberg, A. J. Am. Chem. Soc. 1996, 118, 31683181. (6) Cornelissen, J. J. L. M.; Fischer, M.; Sommerdijk, N. A. J. M.; Nolte, R. J. M. Science 1998, 280, 1427-1430. (7) Sarges, R.; Witkop, B. J. Am. Chem. Soc. 1965, 87, 2011-2020. (8) Fossel, C. T.; Veatch, W. R.; Ovchinnikov, Y. A.; Blout, E. R. Biochemistry 1974, 13, 5264-5275. (9) O’Connell, A. M.; Koeppe, R.; Andersen, O. S. Science 1990, 250, 1256-1259. (10) Zhang, Z.; Pascal, S. M.; Cross, T. A. Biochemistry 1992, 31, 8822-8828. (11) Ketchem, R. R.; Hu, W.; Cross, T. A. Science 1993, 261, 14571460.
Scheme 1. Synthetic Route of g.A-PEG and the Molecular Structure
hydrophobic core of the cell membrane. The hydrophobic peptide was converted to an amphiphilic one by connection of poly(ethylene glycol) (PEG) as a hydrophilic part to the C terminal through an urethane bond.13 The molecular structure and synthetic route is shown in Scheme 1. Three different lengths of PEG, trimer, hexamer, and tridecamer (main fraction), were conjugated with g.A, but only g.A with PEG of tridecamer (g.A-PEG) could be dispersed in aqueous solution. Dynamic light scattering (DLS) measurements of the dispersion showed the formation of aggregates with a diameter of approximately 85 nm, which could be preserved at room temperature for at least 3 days without any change in DLS measurements (the concentration of g.A-PEG was 0.5 mM). The frozenhydrated/cryo-transmission electron microscopy14 (cryoTEM) observation of the g.A-PEG dispersion indicates vesicle formation. The peptide membrane thickness is considered to be less than 5 nm from the dense rim of the unilamellar vesicles in the picture, although the thickness cannot be determined precisely (Figure 1). The unilamellar vesicles are formed by stripping off the skin of multilamellar vesicles by sonication, which is suggested by budding of unilamellar vesicle out of a stuffed particle (12) Chen, Y.; Tucker A.; Wallace, B. A. J. Mol. Biol. 1996, 264, 757769. (13) HCO-Val-Gly-Ala-Leu-Ala-Val-Val-Val-(Trp-Leu)3-Trp-NHCH2CH2-O-CON(CH3)-(CH2CH2O)n-H (n ) 13 on average); g.A was activated by p-nitrophenyl chloroformate and reacted with poly(ethylene glycol) 2-methylaminoethyl ether. FAB mass: 2512 [M + H]+ and several peaks with 44 mass unit (PEG unit) intervals around the main peak. (14) Dubochet, J.; Chang, J. J.; Freeman, R.; Lepault, J.; McDowall, A. W. Ultramicroscopy 1982, 10, 55-62.
10.1021/la981673m CCC: $18.00 © 1999 American Chemical Society Published on Web 05/29/1999
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Figure 3. Temperature dependence of CD spectra of an aqueous dispersion of the g.A-PEG vesicles.
Figure 1. Cryo-TEM picture of the g.A-PEG assembly in water prepared by brief sonication using a probe-type sonicator. Vesicles as well as stuffed particles were observed. The stuffed particles have a multilamellar structure as shown in the inset picture at the right-bottom corner (at the same magnification), which was out of focus to improve the contrast. There are several sites which show formation of unilamellar vesicles by stripping off the outer skin of the multilamellar particles (indicated by arrows).
Figure 2. Effect of Triton X-100 on the encapsulation of FITCPEG in g.A-PEG vesicles. Reference experiment is shown using DMPC liposome.
(multilamellar structure; the inset in Figure 1) as shown by the cryo-TEM picture (Figure 1). An encapsulation experiment was carried out using g.A-PEG aggregates in water. The g.A-PEG vesicles were prepared by using an aqueous solution of fluoresceinlabeled PEG (FITC-PEG; average molecular weight 2000) and dialyzed for 2 days. The dispersion was treated repeatedly by ultrafiltration using the microcon (Amicon,
Figure 4. Illustration of the helix peptide membrane of the g.A-PEG vesicles.
Inc., USA) to remove the free FITC-PEG not encapsulated by the aggregates. The amount of FITC-PEG retained on the filter with the vesicles reached a constant value after the ultrafiltration procedure was repeated several times, which indicates FITC-PEG was encapsulated in the vesicles. The FITC-PEG encapsulated in the vesicles was also shown to be released upon the addition of detergent (Triton X-100) due to the collapse of the vesicles. Collapse of the g.A-PEG vesicles was examined by changing the Triton X-100 concentration. Notably, the g.A-PEG vesicles were stable even at high concentrations of Triton X-100, which destroyed dimyristoylphosphatidylcholine (DMPC) vesicles completely (Figure 2). The hydrophobic core composed of the gramicidin A fragment should be very stable due to the regular packing of the large molecules. Circular dichroism (CD) measurements of a g.A-PEG dispersion show negative Cotton effects at 208 and 228 nm, and a positive effect at 193 nm, indicating an antiparallel double-stranded helix conformation in the aggregates (Figure 3).15-17 The molecular length of the double-stranded helix is agreeable with that estimated from TEM observations. It is therefore considered that the double-stranded helices are packed side-by-side in the g.A-PEG membrane as illustrated in Figure 4. The temperature rise decreased the intensities of the Cotton effect, indicating perturbation in the double-stranded helix (15) Langs, D. A. Science 1988, 241, 188-191. (16) Killian, J. A. Biochim. Biophys. Acta 1992, 1113, 391-425. (17) Chen, Y.: Wallace, B. A. Biophys. J. 1996, 71, 163-170.
Vesicular Self-Assembly of a Helical Peptide
Figure 5. Fluorescence quenching of the aqueous dispersion of the g.A-PEG vesicles by anthracene. An aliquot of an ethanol solution of anthracene was added to the dispersion. The excitation wavelength was 285 nm. Concentrations of g.A-PEG and DMPC were 1 × 10-6 and 1 × 10-4 M, respectively.
at high temperatures. However, the structure appears to be preserved at these temperatures. This structure is interesting in terms of the arrangement of Trp residues. g.A possesses four Trp residues which are located alternately from residues 9 to 15 at the C terminus.
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In the case of the antiparallel double helix of g.A, eight residues are spaced regularly along the double helix axis, where the excited photoenergy should migrate efficiently in the membrane. Indeed, in the presence of anthracene as the photoenergy acceptor, the fluorescence from Trp was quenched more intensively in g.A.-PEG vesicles than in DMPC liposomes under the same acceptor concentrations (Figure 5). Some examples of peptide self-assemblies in water have been reported, such as peptide nanotubes of cyclic peptides18 and β-sheet tapes of a 24-residue amphiphilic peptide to form gels.19 However, it was not certain whether the vesicles with peptides taking a biological motif could be realized. It was believed that higher-molecular-weight amphiphiles with a regular conformation might be difficult to disperse in water because of a higher Kraft point, etc. In the present study, g.A-PEG is dispersed in water forming a unilamellar membrane where the helices are regularly packed. This instance is not considered to be a special case, and other synthetic helical peptides under investigation also adopt the vesicular structure in water. We name the peptide vesicles “peptosome”. Peptosomes will be promising materials for the construction of functional membranes as they provide a regular arrangement of functional groups in the membrane. Acknowledgment. We thank Prof. K. Kaji and Dr. T. Kanaya for the DLS measurements. LA981673M (18) Ghadiri, M. R.; Granja, J. R.; Milligan, R. A.; McRee, D. E.; Hmazanovich, N. Nature 1993, 366, 324-327. (19) Aggeli, A.; Bell, M.; Boden, N.; Keen, J. N.; Knowles, P. F.; McLeish, T. C. B.; Pitkeathly, M.; Radford, S. E. Nature 1997, 386, 259-262.
