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Target-Selective One-Way Membrane Fusion System Based on a pH-Responsive Coiled Coil Assembly at the Interface of Liposomal Vesicles† Ayumi Kashiwada,* Mana Tsuboi, and Kiyomi Matsuda Department of Applied Molecular Chemistry, Graduate School of Industrial Technology, Nihon University, Narashino, Chiba 275-8575, Japan Received September 29, 2010. Revised Manuscript Received October 20, 2010 The coiled coil trimer structure is a common motif observed in membrane fusion processes of specific fusion proteins such as the hemagglutinin glycoprotein. The HA2 subunit in the hemagglutinin changes its conformation or geometry to be favorable to membrane fusion in response to endosomal weakly acidic pH. This pH responsiveness is indispensable to an artificial polypeptide-triggered delivery system as well as the membrane fusion reaction in biology. In this study, we have constructed an AAB-type coiled coil heteroassembled system that is sensitive to weakly acidic pH. The heterotrimer is formed from two kinds of polypeptides containing an Ala or a Trp residue at a hydrophobic a position, and it was observed that the Glu residue at the other a position induced an acidic pH-dependent conformational change. On the basis of this pH-responsive coiled coil heteroassembled system, a boronic acid coupled working polypeptide for the combination of an intervesicular complex with a sugarlike compound on the surface of the target liposome, and a supporting polypeptide for the construction of a pH-responsive heterotrimer with the working polypeptide were designed and synthesized. The process of membrane fusion was characterized by lipid-mixing, inner-leaflet lipid-mixing, and content-mixing assays. The target selective vesicle fusion is clearly observed at a weakly acidic pH, where the working polypeptides form a heterotrimeric coiled coil with the supporting polypeptides in a 1:2 binding stoichiometry and the surfaces between pilot and target vesicles come into close proximity to each other.

Introduction The delivery vehicles in cell-targeting drug or gene delivery systems are required to enter the target cells efficiently. In this process, the plasma membrane and the endosomal membrane exist as large energy barriers. However, it is well known that membrane fusion proteins more easily overcome these barriers in living organisms. Until now, many membrane fusion proteins have been confirmed.1-18 Among these fusion proteins, the viral spike glycoproteins responsible for the penetration of enveloped viruses into their host cells have been well characterized.7-18 † Part of the Supramolecular Chemistry at Interfaces special issue. *Corresponding author. Tel: þ81-47-474-2564. Fax: þ81-47-474-2579. E-mail: [email protected].

(1) S€ollner, T.; Whiteheart, S. W.; Brunner, M.; Erdjument-Bromage, H.; Geromanos, S.; Tempst, P.; Rothman, J. E. Nature 1993, 362, 318–324. (2) Meeusen, S. L.; Nunnari, J. Curr. Opin. Cell Biol. 2005, 17, 389–394. (3) Griffin, E. E.; Detmer, S. A.; Chan, D. C. Biochim. Biophys. Acta 2006, 1763, 482–489. (4) Martin, I.; Epand, R. M.; Ruysschaert, J. M. Biochemistry 1998, 37, 17030– 17039. (5) Jahn, R.; Lang, T.; S€udhof, T. C. Cell 2003, 112, 519–533. (6) Hughson, F. M.; Reinisch, K. M. Curr. Opin. Cell Biol. 2010, 22, 454– 460. (7) S€ollner, T. H. Curr. Opin. Cell Biol. 2004, 16, 429–435. (8) Harrison, S. C. Nat. Struct. Mol. Biol. 2008, 15, 690–698. (9) Lee, J. E.; Fusco, M. L.; Hessell, A. J.; Oswald, W. B.; Burton, D. R.; Saphire, E. O. Nature 2008, 454, 177–182. (10) Roche, S.; Bressanelli, S.; Rey, F. A.; Gaudin, Y. Science 2006, 313, 187– 191. (11) Yin, H. S.; Wen, X.; Paterson, R. G.; Lamb, R. A.; Jardetzky, T. S. Nature 2006, 439, 38–44. (12) Wyatt, R.; Sodroski, J. Science 1998, 280, 1884–1888. (13) Chan, D. C.; Kim, P. S. Cell 1998, 93, 681–684. (14) Bissonnette, M. L. Z.; Donald, J. E.; DeGrado, W. F.; Jardetzky, T. S.; Lamb, R. A. J. Mol. Biol. 2009, 386, 14–36. (15) Carr, C. M.; Kim, P. S. Cell 1993, 73, 823–832. (16) Yu, Y. G.; King, D. S.; Shin, Y. K. Science 1994, 266, 274–276. (17) Bullough, P. A.; Hughson, F. M.; Skehel, J. J.; Wiley, D. C. Nature 1994, 371, 37–43. (18) Kozlov, M. M.; Chemomordik, L. V. Biophys. J. 1998, 75, 1384–1396.

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Influenza virus enters cells by first binding to a sialic acid receptor residing on a target cell surface, followed by endocytosis and finally by an acidic-pH-induced fusion reaction. The fusion protein of the influenza virus is a hemagglutinin (HA) glycoprotein15-18 that contains HA1 and HA2 subunits. The HA1 subunit is responsible for virus binding to sialic acid receptors, and the HA2 subunit harbors a 20-residue hydrophobic fusion polypeptide at the N-terminus, which contributes to the fusion rate. The HA2 subunit forms a nonfusogenic helical-hairpin structure at physiological pH. However, the hairpin region of HA2 rearranges into a fusogenic coiled coil trimer configuration at a weakly acidic pH or in an endosomal environment. This structural rearrangement propels the hydrophobic fusion polypeptide toward the target membrane, leading to membrane fusion. In this way, the pH-responsive coiled coil in the HA2 subunit is considered to play an important role in membrane fusion and viral infection. We think that the coiled coil can be also an attractive structural motif for constructing artificial membrane fusion devices for drug or gene delivery systems. Artificial membrane fusion systems using liposomal vesicles provide an excellent resource for investigating these processes in a quantitative manner under well-controlled conditions. There are some artificial membrane fusion systems controlled by molecular recognition or supramolecular formation at the surfaces of liposomal membranes.19-28 We have also reported one-way (19) Marchi-Artzner, V.; Jullien, L.; Gulik-Krzywicki, T.; Lehn, J. -M. Chem. Commun. 1997, 117–118. (20) Marchi-Artzner, V.; Gulik-Krzywicki, T.; Guedeau-Boudeville, M. -A.; Gosse, C.; Sanderson, J. M.; Dedieu, J. -C.; Lehn, J. -M. ChemPhysChem 2001, 367–376. (21) Ma, M.; Gong, Y.; Bong, D. J. Am. Chem. Soc. 2009, 131, 16919–16926. (22) Richard, A.; Marchi-Artzner, V.; Lalloz, M. N.; Brienne, M. J.; Artzner, F.; Gulik-Krzywicki, T.; Guedeau-Boudeville, M. A.; Lehn, J. M. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 15279–15284. (23) Gong, Y.; Luo, Y.; Bong, D. J. Am. Chem. Soc. 2006, 128, 14430–14431. (24) Gong, Y.; Ma, M.; Luo, Y.; Bong, D. J. Am. Chem. Soc. 2008, 130, 6196– 6205.

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membrane fusion systems toward a sugarlike cys-diol structure on the target liposomes.29,30 In our fusion systems, a well-known molecular recognition pair, phenylboronic acid derivatives and a cyclic cys-diol structure, was used. Although target-selective membrane fusion systems have applications to drug and gene delivery, weakly acidic pH-responsive systems are more suitable and promising systems because the endosomal membranes and tumor tissues are in weakly acidic environments. In terms of the weakly acidic pH-responsiveness, various liposome-based drug-releasing systems have also been designed and constructed.31-36 We select a de novo-designed coiled coil motif as a novel membrane fusion device in this report. De-novo-designed coiled coil polypeptides with pH responsiveness and target selectivity can be easily synthesized according to solid-phase peptide synthesis. Therefore, the coiled coil motif can be used for the construction of artificial membrane fusion devices as mentioned above. There have been a few reports on membrane fusion systems based on coiled coil assemblies, but all of their systems imitate the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-protein-mediated fusion of biological membranes.28,37 In the process of the SNARE-protein-mediated fusion system, the formation of the intercellular (intervesicular) coiled coil assembly is the driving force for fusion. The fusogen made of the SNARE mimetic antiparallel coiled coil provides a simple and effective membrane fusion system, but the SNARE model is not always suitable for constructing a system for specific targets such as glycolipids, glicoproteins, folate receptors, and so on. However, another coiled coil assembly that imitates the pH-sensitive and targetselective HA proteins is expected to display a more effective function as a novel device for target-selective fusion systems. Some reports describe the design of pH-responsive coiled coil polypeptides.38-40 In addition, other reports have discussed coiled coil heteroassemblies bearing more than one functional (25) Stengel, G.; Zahn, R.; H€oo€k, F. J. Am. Chem. Soc. 2007, 129, 9584–9585. (26) Stengel, G.; Simonsson, L.; Campbell, R. A.; H€oo€k, F. J. Phys. Chem. B 2008, 112, 8264–8274. (27) Chan, Y.-H. M.; van Lengerich, B.; Boxer, S. G. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 979–984. (28) Marsden, H. R.; Elbers, N. A.; Bomans, P. H. H.; Sommerdijk, N. A. J. M.; Kros, A. Angew. Chem., Int. Ed. 2009, 48, 2330–2333. (29) Kashiwada, A.; Tsuboi, M.; Matsuda, K. Chem. Commun. 2009, 695–697. (30) Kashiwada, A.; Tsuboi, M.; Mizuno, T.; Nagasaki, T.; Matsuda, K. Soft Matter 2009, 5, 4719–4725. (31) Lee, S. -M.; Chen, H.; Dettmer, C. M.; O’Halloran, T. V.; Nguyen, S. T. J. Am. Chem. Soc. 2007, 129, 15096–15097. (32) Lee, S. -M.; Chen, H.; O’Halloran, T. V.; Nguyen, S. T. J. Am. Chem. Soc. 2009, 131, 9311–9320. (33) Sakaguchi, N.; Kojima, C.; Harada, A.; Kono, K. Bioconjugate Chem. 2008, 19, 1040–1048. (34) Yuba, E.; Kojima, C.; Harada, A.; Tana; Watarai, S.; Kono, K. Biomaterials 2010, 31, 943–951. (35) Cheng, Z.; Chen, A. K.; Lee, H.-Y.; Tsourkas, A. Small 2010, 6, 1398–1401. (36) Pornpattananangkul, D.; Olson, S.; Aryal, S.; Sartor, M.; Huang, C.-M.; Vecchio, K.; Zhang, L. ACS Nano 2010, 4, 1935–1942. (37) Vites, O.; Florin, E. L.; Jahn, R. Biophys. J. 2008, 95, 1295–1302. (38) Suzuki, K.; Yamada, T.; Tanaka, T. Biochemistry 1999, 38, 1751–1756. (39) Dutta, K.; Alexandrov, A.; Huang, H.; Pascal, S. M. Protein Sci. 2001, 10, 2531–2540. (40) Zimenkov, Y.; Dublin, S. N.; Ni, R.; Tu, R. S.; Breedveld, V.; Apkarian, R. P.; Conticello, V. P. J. Am. Chem. Soc. 2006, 128, 6770–6771. (41) Chao, H.; Houston, M. E., Jr.; Grothe, S.; Kay, C. M.; O’Commor-McCourt, M.; Irvin, R. T.; Hodges, R. S. Biochemistry 1996, 35, 12175–12185. (42) Lombardi, A.; Bryson, J. W.; DeGrado, W. F. Biopolymers 1996, 40, 495– 504. (43) Nautiyal, S.; Woolfson, D. N.; King, D. S.; Alber, T. Biochemistry 1995, 34, 11645–11651. (44) Kashiwada, A.; Hiroaki, H.; Kohda, D.; Nango, M.; Tanaka, T. J. Am. Chem. Soc. 2000, 122, 212–215. (45) Schnarr, N. A.; Kennan, A. J. J. Am. Chem. Soc. 2001, 123, 11081–11082. (46) Schnarr, N. A.; Kennan, A. J. J. Am. Chem. Soc. 2002, 124, 9779–9783. (47) Kiyokawa, T.; Kanaori, K.; Tajima, K.; Kawaguchi, M.; Mizuno, T.; Oku, J.; Tanaka, T. Chem.;Eur. J. 2004, 10, 3548–3554.

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polypeptide domain.41-49 Here, we report the design and synthesis of a coiled coil heteroassembly with endosomal (weakly acidic) pH responsiveness in order to construct a pH-responsive domain of a membrane fusion device. Moreover, we discuss the design of a membrane fusion device based on the endosomal-pH-responsive coiled coil heterotrimer with a target-recognizing boronic acid domain and the development of a target-selective membrane fusion system.

Experimental Section Synthesis and Purification of Coiled Coil Polypeptides (IZ-2aW3aE and IZ-2aA). Coiled coil polypeptides IZ-2aW3aE and IZ-2aA were prepared with the standard Fmoc strategy on an Fmoc-Gly-Wang resin (0.61 mmol/g) with a Multi-Syntech Syro XP peptide synthesizer (MultisynTech GmbH, Witten, Germany) on a 0.05 mmol scale by HOBt/TBTU activation. All couplings were performed twice with a 4-fold excess of Fmoc amino acids and coupling reagents. To determine the concentration by UV-vis spectroscopic analysis, the polypeptides were N-terminally labeled with anthranilic acid (Abz). Deprotection and cleavage were performed by treatment with TFA/triisopropylsilane/water (89/10/1, v/v/v) for 2 h. The purification of crude polypeptides was carried out by reverse-phase HPLC on a Knauer smartline manager 5000 system (Knauer GmbH, Berlin, Germany) equipped with a Phenomenex Luna C8 column (10 μm, 250 mm21.2 mm), and the purity was confirmed by analytical HPLC (Phenomenex Luna C8, 5 μm, 250 mm4.6 mm). All products were identified by highresolution ESI-MS (Table S1 in the Supporting Information (SI)).

Synthesis and Purification of Membrane Fusion Device Polypeptides (St-2W3E-BA and St-2A). St-2W3E-BA was synthesized on an Fmoc-Lys(Mtt)-Wang resin (0.61 mmol/g). The Mtt group was removed with a solution of 1% TFA in CH2Cl2. After confirmation of the removal of the Mtt group by a ninhydrin test, the boronic acid derivative30 was coupled to the side-chain amino group of Lys through TBTU/HOBt activation. The reaction was confirmed by the ninhydrin test. The following amino acids were then coupled with the standard Fmoc strategy with a Multi-Syntech Syro XP peptide synthesizer (MultisynTech GmbH, Witten, Germany). To anchor the polypeptide in the liposomal vesicle, stearic acid was coupled to the N-terminus. IZ-2aA was also prepared with the standard Fmoc strategy on an FmocGly-Wang resin (0.61 mmol/g), and stearic acid was coupled to the N-terminus. Deprotection and cleavage were carried out with 2 mL of a solution containing TFA/triisopropylsilane/water (89/10/1, v/v/v) for 2 h. The purification of crude polypeptides was carried out by reverse-phase HPLC on a Knauer Smartline manager 5000 system (Knauer GmbH, Berlin, Germany) equipped with a Phenomenex Jupiter C4 column (10 μm, 250 mm21.2 mm), and the purity was confirmed by analytical HPLC (Phenomenex Jupiter C4, 5 μm, 250 mm  4.6 mm). Polypeptides were eluted with a linear gradient of water/acetonitrile/0.1% TFA. All products were identified by high-resolution ESI-TOF MS using an Agilent 6210 ESI-TOF LC-MS spectrometer (Agilent Technologies Inc., Sanat Clara, CA) (Table S2 in SI). CD Measurements. All CD measurements were performed on a Jasco J-810 spectropolarimeter by the use of a 1-mm-pathlength cuvette at 20 °C (Jasco PTC-348WI peltier thermostat). These measurements were carried out at a total peptide concentration of 30 μM in 10 mM buffer solutions (pH 4.0-6.0, acetic acid/sodium acetate; pH 6.5-7.4, tricine/sodium hydroxide). All buffers contained 100 mM sodium chloride. Concentrations of coiled coil polypeptides were estimated from the UV absorbance of the Abz label at 320 nm. CD spectra were averages of four scans, with data collection from 240 to 190 nm at a 1 nm interval and a 50 nm/min rate. (48) Sakurai, Y.; Mizuno, T.; Hiroaki, H.; Gohda, K.; Oku, J.; Tanaka, T. Angew. Chem., Int. Ed. 2005, 44, 6180–6183. (49) Diss, M. K.; Kennan, A. J. J. Org. Chem. 2008, 73, 9752–9755.

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Kashiwada et al. The binding stoichiometry of the heterotrimer was determined by measuring the mean residue molar ellipticity at 222 nm (θ222) of a series of buffer solutions with a total polypeptide concentration of 80 μM with varying ratios of the IZ-2aW3aE and IZ-2aA polypeptides. Analytical Ultracentrifugation. Sedimentation equilibrium experiments were performed with a Beckman XL-I analytical ultracentrifuge equipped with an An60-Ti rotor. Data were collected with 12-mm-path-length six-sector centerpieces at 320 nm. Polypeptide solutions were prepared at three different concentrations (20, 100, and 200 μM) and rotated with a birefringence in the range of 38 000-48 000 rpm. Each sample solution in rotation is regarded as an equilibrium state when three consecutive scans that are checked every hour become indistinguishable. Solvent densities and partial molar volumes were calculated by Sednterp.62 Data were analyzed with Origin and fit to ideal single-species and appropriate monomer-dimer models. Vesicle Preparation. The vesicles of the following lipid compositions were prepared. The pilot vesicle consists of EggPC, St-2W3E-BA, and St-2A (98.5:0.5:1 mol/mol/mol), whereas EggPC and PI (95:5) constitute the target vesicle. Small unilamellar vesicles (SUVs) were prepared by the evaporation of a chloroform solution of a lipid mixture in a round-bottomed flask, followed by hydration in 10 mM buffer solutions (pH 4.0-6.0, acetic acid/ sodium acetate; pH 6.5-7.4, tricine/sodium hydroxide) containing 100 mM sodium chloride. The suspension was subjected to freezethaw cycles five times to obtain a uniform dispersion. Then, SUVs were prepared for this experiment by the extrusion of the suspension through 100 nm unipore polycarbonate membranes (Whatman) with a mini-extruder set (Avanti). This procedure was performed 10 times, and the final total lipid concentration reached 2.0 mM. Lipid Mixing Assays. Target vesicles bearing NBD-PE and Rh-PE were prepared by the same procedures as described above. The concentrations of NBD-PE and Rh-PE were 0.5 mol % against the lipid mixture. The mixing of phospholipids was followed by the fluorescence resonance energy transfer (FRET) method. The unlabeled (pilot) and labeled (target) vesicles in equal concentrations were mixed with each appropriate buffer solution, and fluorescence values of 531 nm from NBD and 590 nm from Rh were monitored. The degree of fusion was estimated by the lipid mixing defined by I=(It - I0)/(Imax - I0)100, where I0 and It are the fluorescence intensity at 531 nm at time 0 and a defined time (t), respectively, and Imax is the fluorescence (at 531 nm) after the disruption of the vesicles in 0.5% (w/v) Triton X-100. Experimental data were obtained on a Hitachi F-2500 spectrofluorometer at an excitation wavelength of 470 nm. Inner Leaflet Mixing Assays. The reduction of NBD-PEand Rh-PE-labeled vesicles was carried out as follows: A 1:1 mixture of NBD-PE/Rh-PE-labeled vesicles (2.0 mM) and 100 mM sodium dithionite (in a 10 mM tricine/sodium hydroxide buffer solution containing 100 mM sodium chloride) was incubated at 4 °C for 1 h. Free sodium dithionite was removed by gel filtration using Sephadex G-25 fine columns. The rest of the inner leaflet mixing assay was the same lipid mixing assay as described above. Fusion Assay by Contents Mixing. DPX-loaded pilot vesicles and ANTS-loaded target vesicles were prepared by the same procedures as described above and untrapped probes were removed by Sephadex G-25 gel filtration chromatography equilibrated with an assay buffer (10 mM tricine/sodium hydroxide buffer solution containing 100 mM sodium chloride). We used equal concentrations of the pilot (DPX-loaded) and target (ANTSloaded) vesicles diluted in a buffer solution and monitored the fluorescence at 515 nm. The degree of contents mixing was defined by I=(It - I0)/(Iq - I0)100, where I0 and It are the fluorescence intensities at 515 nm at time 0 and at a defined time (t), respectively, and Iq is the fluorescence of quenched vesicle suspensions after the disruption of the vesicles in 0.5% (w/v) Triton X-100. Experimental data were obtained on a Hitachi F-2500 spectrofluorometer at an excitation wavelength of 355 nm. Langmuir 2011, 27(4), 1403–1408

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Liposome Size Distribution Determination. Dynamic light scattering (DLS) of liposome suspensions was studied on an N5 Plus autocorrelator (Beckman-Coulter) equipped with a 25 mW He-Ne laser light source (632.8 nm). Single scans with a 2 min averaging time were performed on the sample at a 90.0° angle. Particle size distributions were calculated from autocorrelation data. All buffer solutions were filtered with a 0.22 μm filter just before liposome preparation. The collection times for the autocorrelation data were 1-4 min. Characterization of the pH-Responsive Behavior of a Designed Membrane Fusion Conjugate in a Liposomal Membrane. The target vesicles bearing Dansyl-PE were prepared by the same procedures as described above. The concentration of Dansyl-PE was 0.5 mol % against the lipid mixture. We measured the fluorescence at 350 nm from Trp and that at 510 nm from Dansyl. The distance between the surface of the pilot vesicle (Trp residue at St-2W3E-BA) and the target vesicle (Dansyl) was estimated by the FRET efficiency (I510/I350). Experimental data were obtained on a Hitachi F-2500 spectrofluorometer at an excitation wavelength of 280 nm.

Results and Discussion Design and Characterization of an Acidic-pH-Responsive Heterotrimeric Coiled Coil. A coiled coil heteroassembly composed of a target-binding and a pH-responsive unit is an attractive model for the design of a target-selective membrane fusion device working in the endosomal environment. We previously designed isoleucine zipper (IZ) polypeptide derivatives containing Ala (IZ-2aA) or Trp (IZ-2aW) at the hydrophobic core a position and demonstrated that these polypeptides form a stable AAB-type (IZ-2aA/ IZ-2aW=2:1) coiled coil heterotrimer.44 The steric matching of 2:1 Ala/Trp core layers in our system is quite important to the formation of the heterotrimer. By taking into account the application of our methodology in constructing a novel coiled coil assembly in response to the acidic pH environment, we have taken an approach to the addition of an acidic-pH-responsive element to the heterotrimeric coiled coil, IZ-2aW3aE, bearing a Glu residue at the hydrophobic 3a position of IZ-2aW (Figure 1). A Glu residue in the hydrophobic core as the macrophage scavenger receptor has been known to play an important role in pH-dependent conformational changes.50 It has also been reported in the de novo-designed coiled coils that the Glu residue mutation at the hydrophobic position leads to a structural change from a random coil at neutral pH to the coiled coil at an acidic pH.38 Thus, it is expected that the novel designed IZ-2aW3aE allows us to form an acidic-pH-dependent heterotrimer through IZ-2aA (IZ-2aA/IZ-2aW3aE = 2:1). Circular dichroism spectroscopy (CD) was used to investigate the secondary structure and folding behavior of the IZ-2aW3aE and IZ-2aA polypeptides. The individual polypeptides show almost the same spectra at pH 7.4 (physiological pH) and pH 5.0 (endosomal pH) (Figure 2a), which indicates their typical random coil conformation. CD spectra of a 2:1 mixture of IZ-2aA and IZ-2aW3aE also show a random coil at pH 7.4 but with typical features of an R-helical secondary structure with two minima at 208 and 222 nm at pH 5.0 (Figure 2b). A plot of pH versus ellipticity at 222 nm (θ222) (Figure 3) also shows that the IZ-2aA/ IZ-2aW3aE mixture forms an acidic-pH-responsive R-helical structure. Furthermore, the ratio of ellipticities (222/208 nm) is greater than 1.0 for the IZ-2aA/IZ-2aW3aE mixture at pH 5.0, which is indicative of the effect due to interactions between R-helices in a coiled coil structure.51,52 This is the first evidence, (50) Suzuki, K.; Doi, T.; Imanishi, T.; Kodama, T.; Tanaka, T. Biochemistry 1997, 36, 15140–15146. (51) Lau, S. Y.; Taneja, A. K.; Hodges, R. S. J. Biol. Chem. 1984, 259, 13253–13261. (52) Cooper, T. M.; Woody, R. W. Biochemistry 1990, 30, 657–676.

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Figure 1. Amino acid sequences of designed polypeptides for constructing AAB-type coiled coil heterotrimers.

Figure 2. CD spectra of a 30 μM solution of the IZ-2aA or IZ-2aW3aE polypeptide individually (a) and a 2:1 mixture of IZ-2aA and IZ-2aW3aE (b). The measurements were performed in 10 mM buffer solutions (buffer = acetic acid/sodium acetate at pH 5.0 and tricine/sodium hydroxide at pH 7.4) containing 100 mM sodium chloride at 20 °C.

Figure 3. Mean residue ellipticity at 222 nm monitored as a function of pH for 30 μM solutions of a 2:1 mixture of IZ-2aA and IZ-2aW3aE. The measurements were made in 10 mM buffer solutions (buffer = acetic acid/sodium acetate at pH 4.0-6.0 and tricine/sodium hydroxide at pH 6.5-7.4) containing 100 mM sodium chloride at 20 °C.

to our knowledge, that pH-responsive heteroassemblies composed of the IZ-2aA and IZ-2aW3aE polypeptides are formed. Analytical ultracentrifugation (AUC) was performed to follow the oligomerization state of the IZ-2aA/IZ-2aW3aE mixture under physiological (pH 7.4) and endosomal (pH 5.0) conditions at three different polypeptide concentrations (20, 100, and 200 μM). A global fit to a single component allowed the determination of the apparent molar mass, which was found to be 3559 ( 109 (monomeric form: the calculated molecular mass for IZ-2aA and IZ-2aW3aE are 3514 and 3646, respectively) at pH 7.4 and 10 805 ( 261 (heterotrimeric form: the calculated molecular mass for (IZ-2aA)2/IZ-2aW3aE is 10 674) at pH 5.0, respectively (Figure S1 in SI). These results reveal that the mixture of IZ-2aA and IZ2aW3aE forms stable heterotrimers at endosomal acidic pH. The binding stoichiometry of the heterotrimer was concomitantly followed by CD spectroscopy. Figure 4 shows a plot of θ222 values as a function of the mole fraction of IZ-2aW3aE for mixtures of 1406 DOI: 10.1021/la103908u

Figure 4. Mean residue ellipticity at 222 nm monitored as a function of the mole fraction of IZ-2aW3aE. The x axis shows the mole fraction titration of IZ-2aW3aE with IZ-2aA at pH 5.0. The total concentration of polypeptides is 30 μM in tricine/sodium hydroxide containing 100 mM sodium chloride at 20 °C.

the IZ-2aW3aE and IZ-2aA polypeptides at pH 5.0, which shows the minimum at a mole fraction of 0.33 for the IZ-2aW3aE polypeptide, corresponding to 2:1 binding stoichiometry. It is evident from these characterization results that the acidicpH-responsive AAB-type coiled coil heterotrimer has been constructed. This newly constructed pH-responsive coiled coil is expected to serve as a major breakthrough leading to the subsequent development of a membrane fusion device working in the endosomal environment. Design of a Novel Membrane Fusion Device Based on an Acidic-pH-Responsive Heterotrimeric Coiled Coil. We have reported that phenylboronic acid derivatives work as effective fusogens toward target liposomal vesicles containing PI.29,30 In particular, the phenylboronic acid derivative with a tertiary amino group in the ortho position (BA1) exhibited rapid vesicle recognition and fusion behavior over a wide pH range, including the endosomal weakly acidic pH range.30 Our goal in this study is to create a novel endosomal-pH-responsive membrane fusion system. Thus, we newly designed a membrane fusion device based on the acidic-pH-responsive coiled coil heterotrimer with a target recognition boronic acid (BA1) domain. On the basis of the results of heterotrimeric coiled coil formation described above, the conjugate of BA1 and the IZ-2aW3aE polypeptide (St-2W3E-BA: Figure 5) was synthesized as a working polypeptide. In addition, the St-2A polypeptide (Figure 5) was also synthesized as a supporting polypeptide for the production of a pH-responsive heterotrimer. The working and supporting polypeptides each had a stearyl group at the N-terminus to anchor the liposomal vesicles. These polypeptides were synthesized by solid-phase peptide synthesis using standard Fmoc chemistry. The key concept of our membrane fusion system is schematically shown in Figure 6. The working and supporting polypeptides in the pilot vesicle exist in random-coil structures over the range of physiological pH. The BA1 domain of the working polypeptide, St-2W3E-BA, forms an intervesicular complex with the inositolcalix on the surface of the target liposome under this condition. However, the BA1 domain contributes to the formation of vesicle cross linking, not to fusion, because of too large a distance between both vesicles. However, the working and supporting polypeptides alike are expected to form heterotrimeric coiled coils at endosomal pH. This coiled coil formation allows the vesicle surfaces to come into close proximity to one another and promotes membrane fusion. Fusogenic Activity. Both the pilot and the target liposomal vesicles used in this study were prepared via a nanoscale technique Langmuir 2011, 27(4), 1403–1408

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Figure 5. Structure of the boronic acid-polypeptide conjugate (working polypeptide: St-2W3E-BA) and a supporting polypeptide (St-2A) as a membrane fusion device with endosomal-pH responsiveness.

Figure 7. FRET experiments for measuring total lipid mixing and inner leaflet lipid mixing. The fluorophores containing target vesicles were mixed with pilot vesicles at pH 7.4 (blue) and 5.0 (red). All of the measurements were performed in 10 mM buffer solutions (buffer = acetic acid/sodium acetate at pH 5.0 and tricine/sodium hydroxide at pH 7.4) containing 100 mM sodium chloride at 30 °C.

Figure 6. Schematic illustration of the pH-responsive membrane fusion system based on our results.

(about 100 nm) in terms of their biocompatibility.53-56 The pilot vesicle contains the novel membrane fusion device (a 1:2 molar ratio of the St-2W3E-BA and St-2A polypeptides), and 1.5 mol % of the devices were anchored in the liposomal membranes composed of EggPC. Vesicle fusion was examined by a probe dilution assay based on the fluorescence resonance energy transfer (FRET) between membrane-bound fluorophores NBD-PE and Rh-PE.57 The FRET fluorophores were incorporated into the target vesicle at an equimolar concentration (0.5 mol %). The extent of membrane fusion was estimated from lipid mixing (%) calculated from the change in the intensity of NBD fluorescence emission. Figure 7 shows the lipid mixing behavior when the pilot vesicles are added to the target vesicles at pH values of 7.4 and 5.0. The device prepared with lipid mixing at pH 7.4 is not fusogenic, judging from the kinetic pattern. However, the kinetics at pH 5.0 indicates that the device works more effectively. The outer leaflet of each vesicle intermixes and so the lipid mixing phenomena always appear, but the inner leaflet does not necessarily participate in the phenomena. To follow the mixing process of the inner leaflet, we have monitored the inner leaflet mixing through the selective reduction of NBD fluorophores in the outer leaflet by sodium dithionite, a membrane-impermeable reductant.58,59 The results of the inner leaflet mixing experiments are shown in Figure 7 as the lipid mixing (%) observed for the (53) Yuan, F.; Leunig, M.; Huang, S. K.; Berk, D. A.; Papahadjopoulos, D.; Jain, R. K. Cancer Res. 1994, 54, 3352–3356. (54) Gerasimov, O. V.; Boomer, J. A.; Qualls, M. M.; Thompson, D. H. Adv. Drug Delivery Rev. 1999, 38, 317–338. (55) Liu, X. -M.; Yang, B.; Wang, Y. -L.; Wang, J. -Y. Chem. Mater. 2005, 17, 2792–2795. (56) Prausnitz, M. R.; Langer, R. Nat. Biotechnol. 2008, 26, 1261–1268. (57) Struck, D. K.; Hoekatra, D.; Pagano, R. E. Biochemistry 1981, 20, 4093– 4099. (58) McIntyre, J. C.; Sleight, R. G. Biochemistry 1991, 30, 11819–11827. (59) Hoekstra, D.; Duzgunes, N. Methods Enzymol. 1993, 220, 15–32.

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dithionite-treated target vesicles blended with the pilot vesicles at pH 5.0. To obtain more information about the fusion events, we have probed the mixing of the aqueous vesicle contents by employing a conventional ANTS/DPX quenching assay.60 Fluorescent ANTS was encapsulated in the target vesicles, and a quencher DPX was encapsulated in the pilot vesicles. The mixing of vesicle contents brought about the fluorescent quenching of ANTS. The pilot vesicles significantly induced internal content mixing at pH 5.0 (Figure 8). The assay of content mixing clearly revealed the membrane fusion process through the dilution of fluorescent lipid probes. Full fusion is accompanied by an increase in the size of the liposome. For the purpose of monitoring the size of the liposome during the membrane fusion process, dynamic light scattering (DLS) measurements were carried out. For a single (pilot or target) vesicle, a significant change in the vesicle size was not observed at pH values of 7.4 and 5.0 for several hours, indicating that vesicle fusion did not occur individually (Figure S2 in SI). DLS experiments were also performed for the equivalent mixture of the pilot (diameter: ca. 110 nm) and target vesicles (diameter: ca. 110 nm). No time-dependent change in the average vesicle diameter was observed in the vesicle size at pH 7.4 (Figure S2 in SI). However, the vesicle size sharply increased and then decreased to around 150 nm at pH 5.0 (Figure S2 in SI). This behavior almost agreed with previous observations.23,29,30 These results show that the target-selective heterotrimeric coiled coil formation from the working and supporting polypeptides under endosomal conditions contributes to effective vesicle fusion, similar to the natural membrane fusion systems mediated by HA glycoprotein. pH-Responsive Behavior of a Designed Device in Liposomal Membranes. To assess whether the pH dependence of fusogenic activity is due to pH-dependent heterotrimeric coiled coil formation in the fusion device, we have measured the circular dichroism (CD) spectra of a 2:1 mixture of St-2A and St-2W3E-BA in EggPC liposome at pH values of 7.4 and 5.0 (Figure S3 in SI). The CD spectra show that the device assumes R-helical structure (60) D€uzg€unes, N.; Wilschut, J. Methods Enzymol. 1993, 220, 3–14.

DOI: 10.1021/la103908u

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pilot and target vesicles plays a crucial role in the vesicular membrane fusion process.27,61 Our results also corroborate that the proposed mechanism of the vesicle fusion system shown in Figure 6 is a reasonable hypothesis.

Conclusions

Figure 8. Contents-mixing assay. ANTS-loaded target vesicles were mixed with DPX-loaded pilot vesicles at pH 7.4 (blue) and 5.0 (red). All of the measurements were made in 10 mM buffer solutions (buffer = acetic acid/sodium acetate at pH 5.0 and tricine/sodium hydroxide at pH 7.4) containing 100 mM sodium chloride at 30 °C.

under acidic conditions. This pH dependence is in good agreement with observation in aqueous solutions (Figure 2). Thus, the pH dependence of the heterotrimeric coiled coil formation in the device can be correlated to the fusogenic activity. To prove our membrane fusion hypothesis shown in Figure 6, we need a large amount of information on the distance between the surfaces of the pilot and the target vesicles in the membrane fusion process. We employed the FRET measurement to evaluate the distance. The St-2W3E-BA polypeptide incorporated into the pilot liposome contains a Trp residue that serves as a fluorescent donor, whereas the PI-bearing target liposome contains the Dansyl fluorophore as a fluorescent acceptor. Noneffective (slight) FRET behavior (I510/I350 =0.203) was observed in the target recognition process at physiological pH (pH 7.4) because of too large a distance between the surfaces of the pilot and the target liposomal vesicles (Figure S4 in SI). The increase in the fluorescence of Dansyl is not remarkable at pH 7.4, whereas control experiments using the free IZ-2aW3aE polypeptide give no FRET behavior (Figure S4 in SI), which supports the formation of an intervesicular complex between the pilot and the target vesicles. However, the excitation of Trp in the St-2W3E-BA polypeptide resulted in an increase in the emission of Dansyl (I510/I350 = 0.532) at endosomal pH (pH 5.0) (Figure S4 in SI). This pH dependence of the FRET behavior shows that the formation of the coiled coil heterotrimer allows the vesicle surfaces to come into close proximity to one another. It is well known that the distance between a

1408 DOI: 10.1021/la103908u

A new class of coiled coil heterotrimers in response to acidic pH has been designed in this study. Our coiled coil assembled system is based on the steric matching of hydrophobic core layers. Moreover, the presence of a Glu residue in the hydrophobic core provides acidic-pH responsiveness to the coiled coil assembly. This coiled coil heteroassembly with acidic-pH responsiveness has the potential to function as a basic component in the construction of future biomaterial-based machines working under endosomal conditions. Thus, to develop a novel target-selective membrane fusion system, we used a fusion device consisting of boronic acid possessing a heterotrimeric coiled coil with acidic-pH responsiveness. The fusion device rapidly and extensively induces endosomalpH-responsive liposomal vesicle fusion, which is correlated with target recognition and pH-responsive coiled coil formation. The application of functional liposomes to a drug or gene delivery system is under investigation. It is possible to develop the delivery vehicle by the use of tailor-made functional liposomes. Our highly effective membrane fusion device has a similar function to the natural membrane fusion systems. Therefore, our designed membrane fusion system will find new opportunities in drug or gene delivery systems. Acknowledgment. We express our appreciation to Prof. M. Hirata for helpful comments on the preparation of the manuscript. Supporting Information Available: Identification of designed polypeptides and polypeptide conjugates. Characterization of the designed polypeptides in aqueous solution. Characterization of the membrane fusion processes by DLS measurements. Structural characterization of the polypeptide device in EggPC liposome. Characterization of the surfaces of the pilot and the target vesicles in the membrane fusion process. This material is available free of charge via the Internet at http://pubs.acs.org. (61) Kasson, P. M.; Kelly, N. W.; Singhal, N.; Vrljic, M.; Brunger, A. T.; Pande, V. S. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 11916–11921. (62) Hayes, D. B.; Laue, T.; Philo, J. Sednterp 1995-1998, University of New Hampshire: Durham, NH.

Langmuir 2011, 27(4), 1403–1408