Vertical and Directional Insertion of Helical Peptide into Lipid Bilayer

Membrane. Koji Nakatani, Tomoyuki Morita, and Shunsaku Kimura*. Department of Material Chemistry, Graduate School of Engineering, Kyoto UniVersity,...
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Langmuir 2007, 23, 7170-7177

Vertical and Directional Insertion of Helical Peptide into Lipid Bilayer Membrane Koji Nakatani, Tomoyuki Morita, and Shunsaku Kimura* Department of Material Chemistry, Graduate School of Engineering, Kyoto UniVersity, Kyoto-Daigaku-Katsura, Nishikyo-ku, Kyoto 615-8510, Japan ReceiVed January 31, 2007. In Final Form: March 23, 2007 A novel helical hexadecapeptide carrying a poly(ethylene glycol) (PEG) chain at the N terminal was synthesized. The N and C terminals of the compound are labeled with a fluorescein isothiocyanate (FITC) group and an N-ethylcarbazolyl group (ECz), respectively. An octapeptide carrying the same groups and a hexadecapeptide without a PEG chain were also synthesized and used as control. A mixture of the peptide and dimyristoylphosphatidylcholine was sonicated in a buffer to prepare the liposome. The orientation as well as direction of the helical segment in the lipid bilayer were analyzed by quenching experiments of the FITC and the ECz fluorescence. The results clearly indicated that the helical segment of the peptide penetrated into the lipid bilayer with vertical orientation in both the gel and liquid crystalline states of the lipid bilayer. Notably, the bulky N terminal was left behind in the outer aqueous phase of liposome, meaning that the C terminal of the peptide points to the inner aqueous phase of liposome. The insertion mode of the helical peptide into a bilayer membrane is therefore well-regulated in terms of the orientation and the directionality by designing the balance between the PEG chain and the helix length. The methodology presented here will initiate a way to construct artificial functional molecular systems that can induce vectorial transport phenomena as seen in biological systems.

Introduction Transmembrane proteins incorporated in biomembranes play an important role in various biological processes such as electron transfer, energy conversion, signal transduction, and so on that are vital for all living organisms.1,2 In most cases, the hydrophobic part of such proteins that traverses a lipid bilayer membrane is regular assemblies constructed by peptide secondary structures. A few of them are made of β-sheets, represented by a β-barrel structure,3 and most of them are made of R-helices. An R-helix is a rigid and cylindrical secondary structure of peptides and is known to form regular self-assemblies via hydrophobic and van der Waals interactions. For example, the photosynthetic reaction center is built by several R-helices that are arranged in a parallel manner to each other with vertical orientation to the membrane. Various chromophores are rationally located in such a rigid framework, and sequential and directional electron-transfer reactions occur among the chromophores to separate charges across the membrane with a quite high quantum yield.4,5 It is also proposed that the R-helix should mediate the long-range electron transfer due to the regular arrangement of the amide groups, and that its large dipole moment formed by accumulation of the amide dipoles may control the direction of the electron transfer.6-12 Natural antimicrobial peptides such as melittin and * Tel: +81-75-383-2400. Fax: +81-75-383-2401. E-mail: shun@ scl.kyoto-u.ac.jp. (1) Lee, A. G. Biochim. Biophys. Acta 2003, 1612, 1-40. (2) Bowie, J. U. J. Mol. Biol. 1997, 272, 780-789. (3) Cowan, S. W.; Garavito, R. M.; Jansonius, J. N.; Jenkins, J. A.; Karlsson, R.; Konig, N.; Pai, E. F.; Pauptit, R. A.; Rizkallah, P. J.; Rosenbusch, J. P.; Rummel, G.; Schirmer, T. Structure 1995, 3, 1041-1050. (4) Deisenhofer, J.; Michel, H. Angew. Chem., Int. Ed. Engl. 1989, 28, 829847. (5) Wasielewski, M. R. Chem. ReV. 1992, 92, 435-461. (6) Hol, W. G. J. Progr. Biophys. Mol. Biol. 1985, 45, 149-195. (7) Galoppini, E.; Fox, M. A. J. Am. Chem. Soc. 1996, 118, 2299-2300. (8) Fox, M. A.; Galoppini, E. J. Am. Chem. Soc. 1997, 119, 5277-5285. (9) Morita, T.; Kimura, S.; Kobayashi, S.; Imanishi, Y. J. Am. Chem. Soc. 2000, 122, 2850-2859. (10) Yasutomi, S.; Morita, T.; Imanishi, Y.; Kimura, S. Science 2004, 304, 1944-1947.

alamethicin are other good examples of formation of a selfassembly of helices spanning through a bilayer membrane.13-17 They have an R-helical segment and are vertically inserted into the hydrophobic region of a lipid bilayer above a critical concentration. They form a regular bundle structure with a pore in the center, and accelerate the ion permeation though the membrane, leading to bacterial lysis. From this point of view, interactions of natural or synthetic model helical peptides with lipid bilayers, their conformation, orientation, and self-assembly formation in the bilayer, have been extensively studied so far.18-32 (11) Sisido, M.; Hoshino, S.; Kusano, H.; Kuragaki, M.; Makino, M.; Sasaki, H.; Smith, T. A.; Ghiggino, K. P. J. Phys. Chem. B 2001, 105, 10407-10415. (12) Morita, T.; Kimura, S.; Kobayashi, S.; Imanishi, Y. Chem. Lett. 2000, 676-677. (13) Shai, Y. Biochim. Biophys. Acta 1999, 1462, 55-70. (14) Wu, M. H.; Maier, E.; Benz, R.; Hancock, R. E. W. Biochemistry 1999, 38, 7235-7242. (15) Huang, H. W. Biochemistry 2000, 39, 8347-8352. (16) Lequin, O.; Ladram, A.; Chabbert, L.; Bruston, F.; Convert, O.; Vanhoye, D.; Chassaing, G.; Nicolas, P.; Amiche, M. Biochemistry 2006, 45, 468-480. (17) Lee, M. T.; Chen, F. Y.; Huang, H. W. Biochemistry 2004, 43, 35903599. (18) Lu, L. P.; Deber, C. M. Biopolymers 1998, 47, 41-62. (19) Bystrom, T.; Strandberg, E.; Kovacs, F. A.; Cross, T. A.; Lindblom, G. Biochim. Biophys. Acta 2000, 1509, 335-345. (20) Fujita, K.; Kimura, S.; Imanishi, Y. Biochim. Biophys. Acta 1994, 1195, 157-163. (21) Otoda, K.; Kimura, S.; Imanishi, Y. Biochim. Biophys. Acta 1992, 1112, 1-6. (22) Smith, M. B.; Tong, J. H.; Genzer, J.; Fischer, D.; Kilpatrick, P. K. Langmuir 2006, 22, 1919-1927. (23) Siegel, D. P.; Cherezov, V.; Greathouse, D. V.; Koeppe, R. E.; Killian, J. A.; Caffrey, M. Biophys. J. 2006, 90, 200-211. (24) Sharpe, S.; Barber, K. R.; Grant, C. W. M.; Goodyear, D.; Morrow, M. R. Biophys. J. 2002, 83, 345-358. (25) Futaki, S. Biopolymers 1998, 47, 75-81. (26) de Planque, M. R. R.; Greathouse, D. V.; Koeppe, R. E.; Schafer, H.; Marsh, D.; Killian, J. A. Biochemistry 1998, 37, 9333-9345. (27) Imanishi, Y.; Kimura, S. Polymer 1996, 37, 4929-4935. (28) Otoda, K.; Kimura, S.; Imanishi, Y. Bull. Chem. Soc. Jpn. 1990, 63, 489-496. (29) Otoda, K.; Kimura, S.; Imanishi, Y. Biochim. Biophys. Acta 1993, 1150, 1-8. (30) Otoda, K.; Kimura, S.; Imanishi, Y. J. Chem. Soc., Perkin Trans. 1 1993, 3011-3016. (31) Kiyota, T.; Lee, S.; Sugihara, G. Biochemistry 1996, 35, 13196-13204.

10.1021/la7002723 CCC: $37.00 © 2007 American Chemical Society Published on Web 05/22/2007

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Figure 1. Chemical structures of the helical peptides (PK16, PK8, and K16) and the control compounds for fluorescence spectroscopy (FITC-PEG and ECz-PEG-ECz).

These studies are important not only for clarifying the structures and functions of natural transmembrane proteins or peptides but also for creating artificial functional molecular systems. In these studies, liposomes that are vesicular bilayer membranes composed of phospholipids have been most frequently used as the model membrane.33-37 There have been many studies focused on vertical incorporation of helical peptides into the bilayer of a liposome. However, to our best knowledge, the directionality of peptides with respect to the membrane plane (toward the inner or outer aqueous phase of the vesicle) has never been controlled. That is because hydrophobic helical peptides tend to form a selfassembly in the lipid bilayer membrane, and once such an assembly is formed, the helical peptides favor antiparallel arrangement to relieve intermolecular dipole-dipole repulsion working between the neighboring peptides.2,38-40 In this study, we aimed at vertical and directional introduction of helical (32) Higashimoto, Y.; Kodama, H.; Jelokhani-Niaraki, M.; Kato, F.; Kondo, M. J. Biochem. 1999, 125, 705-712. (33) Szoka, F.; Papahadjopoulos, D. Annu. ReV. Biophys. Bioeng. 1980, 9, 467-508. (34) Sato, T.; Sunamoto, J. Prog. Lipid Res. 1992, 31, 345-372. (35) Bangham, A. D.; Horne, R. W. J. Mol. Biol. 1964, 8, 660-&. (36) Barenholz, Y. Curr. Opin. Colloid Interface Sci. 2001, 6, 66-77. (37) Kunitake, T.; Okahata, Y. J. Am. Chem. Soc. 1977, 99, 3860-3861. (38) Monera, O. D.; Kay, C. M.; Hodges, R. S. Biochemistry 1994, 33, 38623871. (39) Fujita, K.; Bunjes, N.; Nakajima, K.; Hara, M.; Sasabe, H.; Knoll, W. Langmuir 1998, 14, 6167-6172. (40) Kimura, S.; Muraji, Y.; Sugiyama, J.; Fujita, K.; Imanishi, Y. J. Colloid Interface Sci. 2000, 222, 265-267.

peptides into the liposomal bilayer. Such an asymmetric molecular system is quite interesting for realizing vectorial electron or signal transport across the membrane as seen in biological systems. For this purpose, a hexadecapeptide of an alternating sequence of benzyloxycarbonyl-L-lysine (Lys(Z)) and R-aminoisobutyric acid (Aib) carrying a poly(ethylene glycol) (PEG) chain at the N-terminal was designed (PK16; Figure 1). The Lys(Z)-Aib sequence was chosen for formation of a stable hydrophobic R-helical structure in hydrophobic environment (hydrophobic organic solvents or monolayers).41,42 The hydrophilic PEG chain (polymerization degree of ca. 11) will provide the molecule with an amphiphilic property. On top of that, it will take a globular shape in water so that the PEG segment will be bigger than the diameter of the helical segment, leading to an asymmetric shape. Therefore, it is expected that the peptide will be vertically inserted into the bilayer with the bulky PEG chain left behind in the outer aqueous phase of the liposome, while the peptide C terminal is located closely at the inner aqueous phase; because a liposome has more free space at the outer surface, while it has less free space at the inner surface due to its spherical shape (Figure 2). The N and C terminals of the compound are labeled with a fluorescein isothiocyanate (FITC) group and an N-ethylcarbazolyl (ECz) group, respectively, in order to investigate the peptide (41) Fujita, K.; Kimura, S.; Imanishi, Y.; Okamura, E.; Umemura, J. Langmuir 1995, 11, 1675-1679. (42) Miura, Y.; Kimura, S.; Imanishi, Y.; Umemura, J. Langmuir 1998, 14, 6935-6940.

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Figure 2. Schematic illustration for vertical and directional insertion of PK16 into a DMPC bilayer membrane.

orientation and direction in the bilayer by fluorescence spectroscopy. Dimyristoylphosphatidylcholine (DMPC) was chosen as the component of the liposome. The thickness of the hydrophobic region of the DMPC liposome at the gel state is ca. 30 Å,43 which is not too thick compared to the length of a 16mer R-helical peptide (24 Å).44 On the other hand, the thickness at the liquid-crystalline (LC) state is reported to be 23-25 Å,45 which exactly matches the helix length. Therefore, the helical segment of the molecule should span through the bilayer especially at the LC state of the bilayer if it is vertically inserted (Figure 2). To assess the effects of the peptide length and the PEG chain on the distribution to the liposome, an octapeptide with the same sequence and the same terminal groups and a hexadecapeptide without a PEG chain were used as control (PK8 and K16; Figure 1). The mixture of the peptide and DMPC was sonicated in a buffer solution to prepare the liposome incorporating the peptides in the bilayer. The size of the liposome was characterized by dynamic light scattering (DLS), and the peptide conformation and configuration were investigated by UV-vis absorption, fluorescence, and circular dichroism (CD) spectroscopies. The orientation as well as direction of the helical segment in the bilayer were characterized by determining the locations of the FITC groups and ECz groups, which was carried out by quenching experiments of the FITC and ECz fluorescence with potassium iodide (KI) as a water-soluble quencher. In this measurement, PEG derivatives terminated with a FITC group or two ECz groups were used as the water-soluble reference compounds (FITCPEG and ECz-PEG-ECz; Figure 1). Experimental Section Synthesis of Helical Peptides. PK16, PK8, K16, FITC-PEG, and ECz-PEG-ECz were synthesized by the conventional liquidphase method. The starting octapeptide, Boc-(Lys(Z)-Aib)4-OCH3, was synthesized similarly to the method reported in the literature.46 All the intermediates were identified by 1H NMR spectroscopy, and some of them as well as the final products were further confirmed by FAB or MALDI-TOF mass spectrometry. The purity of each compound was checked by thin-layer chromatography (it generally can detect as little as a few % impurity). The details of the synthesis are available in the Supporting Information. Preparation of Aqueous Dispersion. A chloroform solution of a mixture of the peptide and DMPC (1/200 mol/mol, DMPC 12.5 (43) Tristram-Nagle, S.; Liu, Y. F.; Legleiter, J.; Nagle, J. F. Biophys. J. 2002, 83, 3324-3335. (44) Benedetti, E.; Di Blasio, B.; Pavone, V.; Pedone, C.; Santini, A.; Crisma, M.; Toniolo, C. In Molecular Conformation and Biological Interactions; Balaram, P., Ramaseshan, S., Eds.; Indian Academy of Science: Bangalore, 1991; pp 497-502. (45) Lewis, B. A.; Engelman, D. M. J. Mol. Biol. 1983, 166, 211-217. (46) Otoda, K.; Kitagawa, Y.; Kimura, S.; Imanishi, Y. Biopolymers 1993, 33, 1337-1345.

Nakatani et al. mg) was prepared in a test tube, and the solvent was fully removed by N2 blowing and drying under vacuum to leave a thin film. A Tris-HCl buffer of 3 mL (10 mM, pH 7.4) was added, and the solution was treated by a probe-type sonicator for 5 min at 30 °C, which is above the gel-LC phase transition temperature of a DMPC liposome (24 °C).47 The obtained aqueous dispersion of the DMPC liposome incorporating the peptide was then filtered through a Gelman Sciences Sterile Acrodisc filter (pore size 0.8 µm) to exclude dust mixed in during the preparation. If there was no loss of the compounds by the filtration, the DMPC and peptide concentrations were 6.2 × 10-3 M and 3.1 × 10-5 M, respectively. The prepared liposome dispersion was stored at 30 °C for, at the longest, 3 days until subjected to the measurements. Since there was no significant spectral change during the storage, the aqueous dispersions were considered to be stable. Dynamic Light Scattering Measurements. DLS measurements were carried out to determine the hydrodynamic diameter of the liposome containing the peptide molecules.48 A Brookhaven Instruments BI-200SM light scattering goniometer was used with a vertically polarized incident light of 532 nm from a Spectra-Physics Millennia Pro 2s J Nd:YVO4 laser. The photomultiplier tube was EMI 9893B/350, and the output from it was processed by a Brookhaven Instruments BI-9000AT digital correlator. An electric shutter was attached to the original detector alignment in order to monitor the dark count automatically. The data analysis was performed by the methods reported in literature to estimate the translational diffusion coefficient and thereafter the hydrodynamic diameter.49,50 The peptide concentration was 2.9 × 10-5 M, and the measurement was performed at 20 °C. Spectroscopic Measurements. The UV-vis absorption spectra of the aqueous dispersions were recorded on a Shimadzu UV-2450PC spectrometer at 30 °C with the peptide concentration of 2.5 × 10-5 M. The CD spectra were recorded on a JASCO J-600 CD spectropolarimeter using an optical cell of 0.1 cm optical path length at the peptide concentration of 2.5 × 10-5 M. The data acquisition was performed above the gel-LC transition temperature (24 °C) but at the lowest temperature in which the opaque nature of the dispersion did not disturb the measurements, at 28 °C for the PK8/DMPC and PK16/DMPC dispersions, while at 35 °C for the K16/DMPC dispersion. The fluorescence spectra of the dispersions were recorded on a Hitachi F-4010 fluorometer below and above 24 °C. The excitation wavelengths were 344 and 494 nm for the ECz and FITC fluorescence, respectively. The peptide concentration was set at 9.0 × 10-6 M. The fluorescence quenching and intensity change with time were also carried out by the same fluorometer below and above 24 °C. KI was used as a water-soluble quencher in these experiments.

Results and Discussions The helical peptide derivatives (PK16, PK8, and K16) and the control compounds for fluorescence spectroscopy (FITC-PEG and ECz-PEG-ECz) were synthesized by the conventional liquidphase method. The mixture of the peptide and DMPC (1/200 mol/mol) in a Tris-HCl buffer (pH 7.4) was treated by a probetype sonicator to prepare the liposome incorporating the peptides. It is known that sonication treatment of a liposome dispersion yields small unilamellar vesicles.51,52 During the preparation, the temperature was kept at 30 °C, which is above the gel-LC phase transition temperature of the pure DMPC liposome (Tc ) 24 °C).47 Since PK8 and PK16 have the hydrophilic PEG chain, (47) Harroun, T. A.; Heller, W. T.; Weiss, T. M.; Yang, L.; Huang, H. W. Biophys. J. 1999, 76, 937-945. (48) Osa, M.; Ueda, H.; Yoshizaki, T.; Yaakawa, H. Polym. J. 2006, 38, 153158. (49) Sawatari, N.; Yoshizaki, T.; Yamakawa, H. Macromolecules 1998, 31, 4218-4222. (50) Konishi, T.; Yoshizaki, T.; Yamakawa, H. Macromolecules 1991, 24, 5614-5622. (51) Huang, C. H. Biochemistry 1969, 8, 344 ff. (52) Barenholz, Y.; Gibbes, D.; Litman, B. J.; Goll, J.; Thompson, T. E.; Carlson, F. D. Biochemistry 1977, 16, 2806-2810.

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Figure 4. CD spectra of the peptide/DMPC aqueous dispersions recorded with the peptide concentration of 2.5 × 10-5 M at 28 °C for the PK8/DMPC and PK16/DMPC dispersions and at 35 °C for the K16/DMPC dispersion, respectively.

Figure 3. UV-vis absorption spectra of (a) the peptide/DMPC aqueous dispersions recorded at 30 °C with the peptide concentration of 2.5 × 10-5 M, and (b) reference spectra of the peptides in methanol.

there is a possibility that these compounds can form a unicomponent assembly without DMPC (micelle or bilayer membrane) or can be dissolved in water. However, when PK8 or PK16 were treated in the absence of DMPC, the compound just precipitated and the supernatant showed no absorption from ECz or FITC group, showing that these compounds do not solely form an assembly nor are soluble in water. The hydrodynamic diameters of the pure DMPC, PK16/DMPC, PK8/DMPC, and K16/DMPC liposomes were determined by DLS, respectively, to be 60, 63, 80, and 125 nm. The sizes of the PK16/DMPC and PK8/DMPC liposomes were comparable to that of the pure DMPC liposome, suggesting that these peptides are stably incorporated into the DMPC bilayer due to the amphiphilic property endowed by the PEG chain and do not perturb the original liposome structure. On the other hand, the size of the liposome with K16 that lacks a PEG chain was about twice the size of the pure liposome. It is considered that the K16 peptide can be incorporated into the bilayer, but its surface should become partially hydrophobic due to the fully hydrophobic character of K16, which might facilitate fusion or aggregation of liposomes. To confirm the presence of the peptides in the liposome as well as to get information on the local environment of the FITC

Figure 5. Fluorescence spectra of the peptide/DMPC aqueous dispersions at various temperatures (top) and the fluorescence intensities at various temperatures relative to that at 40 °C (bottom). The fluorescence intensity was the difference between the intensities at 390 and 460 nm as the baseline. The peptide concentration was 9.0 × 10-6 M and the excitation wavelength was 344 nm.

and ECz groups, the aqueous dispersions of the liposomes containing the peptides were subjected to UV-vis absorption spectroscopy at 30 °C. The spectra are shown in Figure 3a. In all the dispersions, an absorption band of the ECz group was observed with the maxima at 240, 278, and 360 nm, although the peak at 344 nm was not clear. For the dispersion containing PK16 or PK8 carrying the PEG chain, an absorption band of the FITC group was also observed at 494 nm along with the shoulder at 464 nm. These results suggested that the peptides were incorporated into the DMPC bilayer. At this stage, it is unknown whether the peptides are inserted into the bilayer membrane or

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Figure 6. Schematic illustrations for incorporation of the peptides into the DMPC bilayer in the gel and LC states.

just physisorbed on the liposome surface. This will be made clear at the later section on fluorescence spectroscopy. The absorption maxima of the ECz group or FITC group in the dispersion agreed with those of a methanol solution of each peptide (Figure 3b), indicating that there is no strong electronic interaction in the ground state among the ECz groups or among the FITC groups in the dispersion. Next, to examine the peptide conformation in the bilayer, CD spectroscopy measurements were performed. The results are shown in Figure 4. Unfortunately, the CD spectrum of the PK8/DMPC dispersion is severely distorted below 220 nm. All the dispersions showed high absorbance due to the scattering and the absorption by the DMPC and buffer solute; on the other hand, the peptide concentration is very low. Therefore, the CD spectra are subject to noise and baseline deviation caused by slight difference between the sample and reference solutions. On top of that, as a short peptide has lower helical content and lower residue concentration, its CD spectrum is more vulnerable to those artifacts compared to that of a long helical peptide. It is thus hard to interpret the conformation of PK8 from the spectrum. On the other hand, for the PK16/DMPC and K16/DMPC dispersions, negative Cotton peaks at 208 and 222 nm were observed, which are characteristic for R-helical conformation. The peak intensity at 222 nm is larger in absolute value than that at 208 nm, suggesting that the R-helical segments form a bundle assembly in the bilayer.53 In comparison to the K16/DMPC dispersion, the PK16/DMPC dispersion has a broad (53) Gibson, N. J.; Cassim, J. Y. Biochemistry 1989, 28, 2134-2139.

Cotton peak at 260-300 nm, which might be due to exciton coupling among the ECz groups because the PK16 peptides are parallel-arranged in the bilayer as discussed later. Fluorescence from the ECz groups was analyzed with varying temperature. The fluorescence spectra are shown in the top panel of Figure 5. Monomer emission (370 and 390 nm) without excimer emission was observed in all the dispersions at all the temperatures. It is well-known that an ECz group can hardly form an excimer even at high concentrations of the chromophore due to the bulky substitute on the N atom.54,55 However, the fluorescence intensities were quite different from peptide to peptide in the bilayer. The most intensive emission was observed in the K16/ DMPC dispersion, while that in the PK16/DMPC dispersion was about half and that in the PK8/DMPC dispersion was the lowest at each temperature from 15 to 40 °C. Note that the vertical axis has a different range in each spectrum. In general, there are two factors which explain the change of fluorescence intensity: local environment polarity and concentration quenching. On the basis of the latter reason, it is speculated that the ECz groups may be separated well in the K16/DMPC bilayer by forming a bundle structure in an antiparallel arrangement of helices, while they are close to each other in the PK16/DMPC and PK8/DMPC bilayers with that in a parallel arrangement (Figure 6). As the K16 peptide lacks a PEG chain, its direction in the bundle (54) Morita, T.; Kimura, S.; Imanishi, Y. J. Phys. Chem. B 1997, 101, 45364538. (55) Morita, T.; Kimura, S.; Imanishi, Y. J. Am. Chem. Soc. 1999, 121, 581586.

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Figure 7. Stern-Volmer plots for the quenching experiments of the peptide/DMPC aqueous dispersions using KI as the quencher for the FITC (top) and ECz (bottom) fluorescence at 17 (right) and 31 °C (left). The excitation wavelengths were 494 and 344 nm for the FITC and ECz fluorescence, respectively.

assembly may not be controlled, and an antiparallel arrangement of the neighboring helical segments in the bilayer should prevail to relieve the dipole-dipole repulsion between them. The locations of the ECz groups are separated up and down with respect to the bilayer (Figure 6e,f). On the other hand, the peptide with a PEG chain should be preferentially inserted into the bilayer with the bulky PEG chain in the outer aqueous phase, leading to a parallel arrangement of the helical segments in which the ECz groups are close to each other (Figure 6a-d). These speculations based on the chemical structures of the peptides agree well with the observed trend in the emission intensities of the ECz groups. However, the reason for the faint emission of the PK8/DMPC dispersion is yet to be clarified. Intramolecular energy transfer from the ECz group to the FITC group in the shorter separation may be considered another reason. One might consider that the bulky PEG chain would prevent bundling of the helices. However, on the basis of the results presented here, the PEG group should be flexible enough to allow the peptides to form a self-assembly. The emission intensities at 390 nm at various temperatures relative to that at 40 °C in each dispersion are shown in the bottom panel of Figure 5. Note that the intensity difference between 390 and 460 nm was plotted considering the baseline shift. Qualitatively, a similar behavior was observed in all the peptides in the bilayer. The low intensity at lower temperatures abruptly increases beyond the Tc (24 °C) and then slightly decreases with increasing temperature. The low intensity below the Tc is reasonably explained by concentration quenching due to the peptide association in the bilayer, which is a general observation of phase separation in peptide-lipid bilayer mixture

at temperatures below the Tc.56 On the other hand, above the Tc, the lipids are so fluidic that they accommodate each other to make the peptides disperse in the bilayer. However, smaller clusters of PK16 or K16 peptide than those below the Tc should be formed even in the bilayer above the Tc as suggested by CD spectroscopy (Figure 6b,d,f). The gradual decrease of the emission with temperature increase above the Tc is general and due to the thermal activation of an irradiative deactivation process of the excited state of the chromophore. The orientation and direction of the helical segments in the bilayer were clarified by fluorescence quenching of FITC and ECz fluorophores with using KI below and above the Tc. KI has been widely used as a water-soluble fluorescence quencher for analysis of biochemical systems.57,58 An aliquot of KI was added successively to the dispersion, and the fluorescence changes were recorded. The quencher is considered to be distributed only in the outer aqueous phase of the liposome system, and in some cases, it slowly moves into the inner aqueous phase via diffusion through the bilayer. The transfer of KI across the membrane is discussed later. The Stern-Volmer plots for the FITC fluorescence quenching of the PK16/DMPC and PK8/DMPC dispersions and the FITCPEG aqueous solution are shown in the top panel of Figure 7. At 17 °C, the quenching behaviors of the PK16/DMPC and PK8/ (56) Uemura, A.; Kimura, S.; Imanishi, Y. Biochim. Biophys. Acta 1983, 729, 28-34. (57) Rachofsky, E. L.; Seibert, E.; Stivers, J. T.; Osman, R.; Ross, J. B. A. Biochemistry 2001, 40, 957-967. (58) Ajtai, K.; Ilich, P. J. K.; Ringler, A.; Sedarous, S. S.; Toft, D. J.; Burghardt, T. P. Biochemistry 1992, 31, 12431-12440.

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Figure 8. Time course of the change in the fluorescence intensity of the FITC (top) and ECz (bottom) fluorescence in the peptide/DMPC aqueous dispersions at 17 (right) and 31 °C (left) with addition of KI.

DMPC dispersions are the same as that of the FITC-PEG solution as control, showing that all the FITC groups of the peptides are located in the outer aqueous phase of the liposome. On the other hand, at 31 °C, the quenching occurs comparably between the PK8/DMPC dispersion and the FITC-PEG solution, but it is slightly inhibited in the PK16/DMPC dispersion. This finding may suggest that, on the basis of the quenching ratios of the FITC groups, nearly 20% of the FITC groups of PK16 are located in the inner aqueous phase. A part of the peptides may flip-flop, aided by the fluidity of the membrane above Tc, to relieve strong intermolecular dipole-dipole repulsion between parallel-arranged helices. This point is yet to be cleared. Taken together, it can be concluded that a majority of the FITC groups of the PK16 or PK8 peptide are in the outer aqueous phase; in other words, the peptides are asymmetrically introduced into the bilayer leaving the PEG chain in the outer aqueous phase both below and above the Tc (Figure 6a-d). On the other hand, the ECz fluorescence was hardly quenched (Figure 7 bottom). At 17 °C, the quenching in the PK8/DMPC and K16/DMPC dispersions is significantly low, and the fluorescence of the PK16/DMPC dispersion is nearly unchanged even at the high concentration of quencher, even though the fluorescence of ECz-PEG-ECz as control was effectively quenched by KI. This result shows that only a small portion of the ECz groups of PK8 or K16 are located at the outer phase, while nearly all the ECz groups of PK16 are shielded from the access of quencher in the outer aqueous phase. Interestingly, the partial quenching in the PK8/DMPC dispersion was changed to no quenching when the temperature was raised to 31 °C. This observation suggests that the ECz groups of PK8 partially located near the outer aqueous phase should be transferred toward the inner aqueous phase with the phase transition of the bilayer from

the gel to the LC state. It has been demonstrated that a fluid lipid bilayer in the LC state can adjust its hydrophobic thickness to match the length of incorporated hydrophobic peptides to minimize the hydrophobic area exposed to water.26,47,59 It is thus considered that the rigid DMPC bilayer in the gel state cannot accommodate the PK8 peptides suitably due to the mismatch in its thickness with the helical length, leading to irregular arrangement of the helical segments including horizontal orientation on the membrane surface (Figure 6c). In the LC state, the flexible bilayer can shrink to conform to the shorter helical segments of PK8 with a vertical orientation (Figure 6d). On the other hand, the ECz groups in the K16/DMPC dispersion were quenched by KI both below and above the Tc. Comparing the quenching in the K16/DMPC dispersion at 31 °C with that in the ECz-PEG-ECz solution as control, we see that nearly half of the ECz groups of K16 are exposed to the outer aqueous phase. This finding also supports the antiparallel arrangement of the K16 peptides in the bilayer with vertical orientation (Figure 6f). The lower quenching degree observed at 17 °C may imply formation of large aggregates of K16 on the membrane surface, in which some of the ECz groups are buried in and not accessible from the outer aqueous phase. During the quenching experiments, we noticed that the fluorescence intensity decreased immediately after the addition of the quencher, but it gradually decayed over a long period in some cases. This slow process is considered due to the quenching of the chromophores exposed to the inner aqueous phase by slow diffusion of the quencher into the inner aqueous phase through the bilayer. The time courses of the fluorescence intensity changes of the FITC and ECz groups in the dispersions with the quencher (59) Kandasamy, S. K.; Larson, R. G. Biophys. J. 2006, 90, 2326-2343.

Directional Insertion of Peptide into Lipid Bilayer

Langmuir, Vol. 23, No. 13, 2007 7177

addition taken as time zero are shown in Figure 8. For the FITC emission in the PK8/DMPC and PK16/DMPC dispersions (Figure 8 top), the fluorescence was quenched immediately after the addition. The subsequent slow quenching was observed only in the PK8/DMPC dispersion at 31 °C. The majority of the FITC groups are exposed to the outer aqueous phase, which interpretation is in agreement with the results of the Stern-Volmer plot analysis. On the other hand, the ECz emission (Figure 8 bottom) in the cases of the PK16/DMPC dispersion at 17 and 31 °C, and the PK8/DMPC dispersion at 31 °C showed just faint or no quenching immediately after the KI addition, showing that the ECz groups are located at the inner phase and there is no defect in the bilayer allowing the quencher to diffuse through (Figure 6a,b,d). It seems that the PK16 peptides are well-packed with the DMPC bilayer even in the gel state despite the slight mismatch between the helical length (24 Å) and the bilayer hydrophobic thickness (30 Å). Meanwhile, the PK8/DMPC dispersion at 17 °C showed gradual quenching after the KI addition, suggesting defect formation in the bilayer. This result is reasonable because the packing of the PK8 peptides with the rigid DMPC bilayer is considered poor, as explained above. On the other hand, in the K16/DMPC dispersion at 17 and 31 °C, both quick quenching for the chromophores at the outer phase and slow quenching for those at the inner phase were observed, indicative of the defect formation in the bilayer due to the insertion of the peptide without amphiphilicity. Furthermore, the degrees of both quenching processes were comparable especially at 31 °C (indicated by dotted lines). This finding further supports the antiparallel arrangement of the K16 peptides that penetrate through the bilayer. Taken together, it is finally concluded that the PK16 peptides are inserted into the DMPC bilayer in both the gel and LC states with a hydrophilic PEG chain protruding into the outer aqueous phase, and the peptides form a large-sized assembly below Tc while they are more dispersed in the bilayer as a smaller assembly above Tc.

orientation and directionality with the PEG chain protruding into the outer aqueous phase of the liposome in both the gel and LC states of the bilayer. By comparison with the control peptide with a shorter helix length or lacking a PEG chain, it was indicated that the matching between the helix length and the thickness of the hydrophobic region of the bilayer and the presence of the PEG chain that makes the molecule amphiphilic and increases the molecular curvature are essential for such vertical and directional introduction of helical peptides into a lipid bilayer membrane. As briefly mentioned in the Introduction, such an asymmetric membrane system is interesting for the realization of directional transports of energy or mass across the membrane, which are vital functions of biomembranes. For example, vectorial electron-transfer reactions across the membrane are attractive from the viewpoint of the natural photosynthetic systems. The ECz group used in this study, located at the inner aqueous phase, can be utilized as a photosensitizer to trigger photoinduced electron transfer across the membrane. As the dipole moment is directed from the inner to outer phase in the system, vectorial electron transfer from the inner to outer phase would be facilitated by the dipole moment upon photoexcitation of the ECz group, in the presence of an electron donor and acceptor in the inner and outer aqueous phases, respectively.10,12 Such a vectorial electron-transfer system across the membrane as a simple model of electron transports in biological systems is now under investigation.

Conclusion

Supporting Information Available: Details of the synthesis of the compounds. This material is available free of charge via the Internet at http://pubs.acs.org.

In this study, we demonstrated that a hydrophobic R-helical peptide carrying a hydrophilic PEG chain at the terminal is stably incorporated into a DMPC bilayer membrane with vertical

Acknowledgment. This work is partly supported by Grantin-Aids for Young Scientists B (16750098), for Exploratory Research (17655098), and for Scientific Research B (15350068), and 21st century COE program, COE for a United Approach to New Materials Science, from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. The authors would like to thank Prof. Yoshizaki and Dr. Osa of Department of Polymer Chemistry in Kyoto University for their kind help in the dynamic light scattering measurements.

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