Biological Identification of Peptides that Specifically Bind to Poly

Oct 15, 2010 - Kacar, T.; Parviz, B. A.; Sarikaya, M. Small 2006, 2, 1372–1378. (22) Hnilova, M.; Oren, E. E.; Seker, U. O. S.; Wilson, B. R.; Colli...
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Biological Identification of Peptides that Specifically Bind to Poly(phenylene vinylene) Surfaces: Recognition of the Branched or Linear Structure of the Conjugated Polymer Hirotaka Ejima,† Hisao Matsuno,‡, and Takeshi Serizawa*,§

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† Department of Chemistry and Biotechnology, Graduate School of Engineering, ‡Komaba Open Laboratory (KOL), and §Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan. Current address: Department of Applied Chemistry, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan

Received May 19, 2010. Revised Manuscript Received September 23, 2010 Peptides that bind to poly(phenylene vinylene) (PPV) were identified by the phage display method. Aromatic amino acids were enriched in these peptide sequences, suggesting that a π-π interaction is the key interaction between the peptides and PPV. The surface plasmon resonance (SPR) experiments using chemically synthesized peptides demonstrated that the Hyp01 peptide, with the sequence His-Thr-Asp-Trp-Arg-Leu-Gly-Thr-Trp-His-His-Ser, showed an affinity constant (7.7  105 M-1) for the target, hyperbranched PPV (hypPPV) film. This value is 15-fold greater than its affinity for linear PPV (linPPV). In contrast, the peptide screened for linPPV (Lin01) showed the reverse specificity for linPPV. These results suggested that the Hyp01 and Lin01 peptides selectively recognized the linear or branched structure of PPVs. The Ala-scanning experiment, circular dichroism (CD) spectrometry, and molecular modeling of the Hyp01 peptide indicated that adequate location of two Trp residues by forming the polyproline type II (PII) helical conformation allowed the peptide to specifically interact with hypPPV.

Introduction Molecular recognition processes such as receptor-ligand, antigen-antibody, or sugar-lectin interactions play pivotal roles in biological systems. Recently, it has become increasingly clear that the specific targets of biomolecules are not necessarily typical biomolecules. For instance, Zheng and co-workers reported singlestrand DNAs that recognize the chiralities of carbon nanotubes (CNTs) and used them to isolate semiconductive CNTs.1,2 Lerner and co-workers screened anti-stilbene antibodies,3,4 and Janda et al. used them as a sensor for mercury.5 Belcher and co-workers developed high-power lithium-ion batteries using filamentous viruses displaying peptides that have high affinity with inorganic electrode materials.6,7 However, it is normally difficult to rationally design such unique biomolecules potentially utilized in materials science. The most promising methods to practically identify biomolecules that recognize artificial materials rely upon an affinity-based *To whom correspondence should be addressed. Telephone: þ81-3-54525224. Fax: þ81-3-5452-5224. E-mail: [email protected]. (1) Zheng, M.; Jagota, A.; Strano, M. S.; Santos, A. P.; Barone, P.; Chou, S. G.; Diner, B. A.; Dresselhaus, M. S.; McLean, R. S.; Onoa, G. B.; Samsonidze, G. G.; Semke, E. D.; Usrey, M.; Walls, D. J. Science 2003, 302, 1545–1548. (2) Tu, X. M.; Manohar, S.; Jagota, A.; Zheng, M. Nature 2009, 460, 250–253. (3) Simeonov, A.; Matsushita, M.; Juban, E. A.; Thompson, E. H. Z.; Hoffman, T. Z.; Beuscher, A. E.; Taylor, M. J.; Wirsching, P.; Rettig, W.; McCusker, J. K.; Stevens, R. C.; Millar, D. P.; Schultz, P. G.; Lerner, R. A.; Janda, K. D. Science 2000, 290, 307–313. (4) Debler, E. W.; Kaufmann, G. F.; Meijler, M. M.; Heine, A.; Mee, J. M.; Pljevaljcic, G.; Di Bilio, A. J.; Schultz, P. G.; Millar, D. P.; Janda, K. D.; Wilson, I. A.; Gray, H. B.; Lerner, R. A. Science 2008, 319, 1232–1235. (5) Matsushita, M.; Meijler, M. M.; Wirsching, P.; Lerner, R. A.; Janda, K. D. Org. Lett. 2005, 7, 4943–4946. (6) Nam, K. T.; Kim, D. W.; Yoo, P. J.; Chiang, C. Y.; Meethong, N.; Hammond, P. T.; Chiang, Y. M.; Belcher, A. M. Science 2006, 312, 885–888. (7) Lee, Y. J.; Yi, H.; Kim, W. J.; Kang, K.; Yun, D. S.; Strano, M. S.; Ceder, G.; Belcher, A. M. Science 2009, 324, 1051–1055. (8) Hoogenboom, H. R.; de Bruine, A. P.; Hufton, S. E.; Hoet, R. M.; Arends, J. W.; Roovers, R. C. Immunotechnology 1998, 4, 1–20.

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selection of desired biomolecules from combinatorially constructed biomolecular libraries.8,9 Regarding material-binding peptides, phage display10 and cell-surface display11 methods have been commonly utilized.12-14 Based on these methodologies, peptides that specifically bind to metal oxides,15,16 metals,17-25 semiconductors,26,27 magnetics,28 nanocarbons,29-33 and polymers34-43 (9) Wilson, D. S.; Szostak, J. W. Annu. Rev. Biochem. 1999, 68, 611–647. (10) Smith, G. P. Science 1985, 228, 1315–1317. (11) Wittrup, K. D. Curr. Opin. Biotechnol. 2001, 12, 395–399. (12) Sarikaya, M.; Tamerler, C.; Jen, A. K. Y.; Schulten, K.; Baneyx, F. Nat. Mater. 2003, 2, 577–585. (13) Sarikaya, M.; Tamerler, C.; Schwartz, D. T.; Baneyx, F. O. Annu. Rev. Mater. Res. 2004, 34, 373–408. (14) Baneyx, F.; Schwartz, D. T. Curr. Opin. Biotechnol. 2007, 18, 312–317. (15) Brown, S. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 8651–8655. (16) Sano, K. I.; Shiba, K. J. Am. Chem. Soc. 2003, 125, 14234–14235. (17) Brown, S. Nat. Biotechnol. 1997, 15, 269–272. (18) Naik, R. R.; Stringer, S. J.; Agarwal, G.; Jones, S. E.; Stone, M. O. Nat. Mater. 2002, 1, 169–172. (19) Brown, S.; Sarikaya, M.; Johnson, E. J. Mol. Biol. 2000, 299, 725–735. (20) Tamerler, C.; Oren, E. E.; Duman, M.; Venkatasubramanian, E.; Sarikaya, M. Langmuir 2006, 22, 7712–7718. (21) Tamerler, C.; Duman, M.; Oren, E. E.; Gungormus, M.; Xiong, X. R.; Kacar, T.; Parviz, B. A.; Sarikaya, M. Small 2006, 2, 1372–1378. (22) Hnilova, M.; Oren, E. E.; Seker, U. O. S.; Wilson, B. R.; Collino, S.; Evans, J. S.; Tamerler, C.; Sarikaya, M. Langmuir 2008, 24, 12440–12445. (23) So, C. R.; Tamerler, C.; Sarikaya, M. Angew. Chem., Int. Ed. 2009, 48, 5174–5177. (24) Seker, U. O. S.; Wilson, B.; Sahin, D.; Tamerler, C.; Sarikaya, M. Biomacromolecules 2009, 10, 250–257. (25) Kacar, T.; Zin, M. T.; So, C.; Wilson, B.; Ma, H.; Gul-Karaguler, N.; Jen, A. K. Y.; Sarikaya, M.; Tamerler, C. Biotechnol. Bioeng. 2009, 103, 696–705. (26) Whaley, S. R.; English, D. S.; Hu, E. L.; Barbara, P. F.; Belcher, A. M. Nature 2000, 405, 665–668. (27) Lee, S. W.; Mao, C. B.; Flynn, C. E.; Belcher, A. M. Science 2002, 296, 892– 895. (28) Reiss, B. D.; Mao, C. B.; Solis, D. J.; Ryan, K. S.; Thomson, T.; Belcher, A. M. Nano Lett. 2004, 4, 1127–1132. (29) Wang, S. Q.; Humphreys, E. S.; Chung, S. Y.; Delduco, D. F.; Lustig, S. R.; Wang, H.; Parker, K. N.; Rizzo, N. W.; Subramoney, S.; Chiang, Y. M.; Jagota, A. Nat. Mater. 2003, 2, 196–200. (30) Kase, D.; Kulp, J. L.; Yudasaka, M.; Evans, J. S.; Iijima, S.; Shiba, K. Langmuir 2004, 20, 8939–8941.

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have been identified so far. For instance, Belcher et al. obtained chlorine-doped polypyrrole-binding peptides for polymersurface functionalization36 and semiconductor-binding peptides for nanocrystal assembly.26 Sarikaya and co-workers reported gold-binding peptides19-25 with specificity over another noble metal, platinum,21 and used them for enzyme immobilization on gold substrates.25 Our group also reported peptides that successfully recognize the stereoregularity38-41 and crystallinity42 of commodity polymers with simple chemical structures. The adsorption of a model protein fused with an isotactic poly(methyl methacrylate) (it-PMMA)-binding peptide, c02, was specifically enhanced toward the it-PMMA film surface.43 Thus, exploring specific combinations between biomolecules and artificial materials opens up novel functions and potential applications for both the materials and biomolecules. However, quantitative assessments of the binding specificity of biomolecules toward sufficiently analogous materials are still limited despite the central and essential science of molecular recognition. Conjugated polymers are attractive artificial materials with intrinsic optical and electronic properties, and they have been utilized in a number of applications which include light emitting diodes,44 photovoltaic cells,45 and biosensing.46 A representative conjugated polymer, poly(phenylene vinylene) (PPV), which has a simple chemical structure consisting of phenyl rings and vinyl groups, is the most extensively studied conjugated polymer as synthetic target. The development of precisely controlled syntheses allowed us to obtain stereocontrolled,47,48 regioregular,49,50 and dendritic PPV.51,52 Recently, dendritic PPVs, including dendrimers and hyperbranched polymers, are gaining increasing attention due to their advantageous solution processing (31) Su, Z. D.; Leung, T.; Honek, J. F. J. Phys. Chem. B 2006, 110, 23623–23627. (32) Su, Z.; Mui, K.; Daub, E.; Leung, T.; Honek, J. J. Phys. Chem. B 2007, 111, 14411–14417. (33) Zheng, L. F.; Jain, D.; Burke, P. J. Phys. Chem. C 2009, 113, 3978–3985. (34) Adey, N. B.; Mataragnon, A. H.; Rider, J. E.; Carter, J. M.; Kay, B. K. Gene 1995, 156, 27–31. (35) Berglund, J.; Lindbladh, C.; Nicholls, I. A.; Mosbach, K. Anal. Commun. 1998, 35, 3–7. (36) Sanghvi, A. B.; Miller, K. P. H.; Belcher, A. M.; Schmidt, C. E. Nat. Mater. 2005, 4, 496–502. (37) Watanabe, H.; Tsumoto, K.; Taguchi, S.; Yamashita, K.; Doi, Y.; Nishimiya, Y.; Kondo, H.; Umetsu, M.; Kumagai, I. Bioconjugate Chem. 2007, 18, 645–651. (38) Serizawa, T.; Sawada, T.; Matsuno, H.; Matsubara, T.; Sato, T. J. Am. Chem. Soc. 2005, 127, 13780–13781. (39) Serizawa, T.; Sawada, T.; Kitayama, T. Angew. Chem., Int. Ed. 2007, 46, 723–726. (40) Serizawa, T.; Sawada, T.; Matsuno, H. Langmuir 2007, 23, 11127–11133. (41) Serizawa, T.; Techawanitchai, P.; Matsuno, H. ChemBioChem 2007, 8, 989–993. (42) Matsuno, H.; Sekine, J.; Yajima, H.; Serizawa, T. Langmuir 2008, 24, 6399– 6403. (43) Matsuno, H.; Date, T.; Kubo, Y.; Yoshino, Y.; Tanaka, N.; Sogabe, A.; Kuroita, T.; Serizawa, T. Chem. Lett. 2009, 38, 834–835. (44) Burroughes, J. H.; Bradley, D. D. C.; Brown, A. R.; Marks, R. N.; Mackay, K.; Friend, R. H.; Burns, P. L.; Holmes, A. B. Nature 1990, 347, 539–541. (45) Yu, G.; Gao, J.; Hummelen, J. C.; Wudl, F.; Heeger, A. J. Science 1995, 270, 1789–1791. (46) Feng, F. D.; He, F.; An, L. L.; Wang, S.; Li, Y. H.; Zhu, D. B. Adv. Mater. 2008, 20, 2959–2964. (47) Katayama, H.; Nagao, M.; Moriguchi, R.; Ozawa, F. J. Organomet. Chem. 2003, 676, 49–54. (48) Katayama, H.; Nagao, M.; Nishimura, T.; Matsui, Y.; Umeda, K.; Akamatsu, K.; Tsuruoka, T.; Nawafune, H.; Ozawa, F. J. Am. Chem. Soc. 2005, 127, 4350–4353. (49) Pan, M.; Bao, Z. N.; Yu, L. P. Macromolecules 1995, 28, 5151–5153. (50) Suzuki, Y.; Hashimoto, K.; Tajima, K. Macromolecules 2007, 40, 6521– 6528. (51) Deb, S. K.; Maddux, T. M.; Yu, L. P. J. Am. Chem. Soc. 1997, 119, 9079– 9080. (52) Meier, H.; Lehmann, M. Angew. Chem., Int. Ed. 1998, 37, 643–645. (53) Ejima, H.; Iwata, T.; Yoshie, N. Macromolecules 2008, 41, 9846–9848. (54) Lim, S. J.; Seok, D. Y.; An, B. K.; Jung, S. D.; Park, S. Y. Macromolecules 2006, 39, 9–11.

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Figure 1. Selection of PPV-binding peptides. (a) Chemical structures of the target polymers, hypPPV and linPPV. (b) Schematic illustration of the selection for PPV using a random 12-mer peptide library.

properties,53 tunable emission color,54 efficient intramolecular electron transfer,55 and so on. However, biological applications of the dendritic conjugated polymers other than linear counterparts are sparse56 in spite of their advantages. In order to utilize the polymers in biologically oriented applications, a molecular-level understanding and the constructive design of specific interactions between the polymers and biomolecules are strongly desirable. Specifically binding biomolecules for the conjugated polymers would open the door to new ways of dispersing the polymers in aqueous phase and developing novel biosensing systems based on the change in the polymer fluorescence. Here, we identified 12-mer peptides that specifically and strongly bound to PPV surfaces based on affinity selections, socalled biopanning, from a phage-displayed peptide library. We employed not only normal linPPV but also hypPPV as the distinctive targets for the identified peptides (Figure 1a). The bindings of isolated phage clones and subsequently synthetic peptides to each PPV surface were analyzed quantitatively by titering and surface plasmon resonance (SPR) measurements. The identified peptides for each target showed excellent specificity, recognizing the slight structural difference in the PPV isomers, that is, branched or linear structures. Mutation and spectral analysis of an excellent peptide suggested the essential amino acids and backbone conformation. This paper is the first demonstration to reveal novel peptides that significantly recognize a delicate difference in conjugated polymers.

Experimental Section Film Preparation. Hyperbranched PPV (hypPPV) was

synthesized according to a previously reported method.54 The weight-average molecular weight (Mw) and polydispersity index (PDI) of hypPPV, determined by gel permeation chromatography (GPC) using polystyrene standards, were 1700 and 1.2, respectively. A precursor of linear PPV (linPPV), poly(p-xylene tetrahydrothiophenium chloride) was purchased from Aldrich. For SPR measurements, the PPV films were prepared on Au-coated glass (SIA Kit Au, GE Healthcare). CHCl3 solutions of hypPPV (1.0 mg mL-1) were spin-coated for 60 s at a scan rate of 2000 rpm. Aqueous solutions of the linPPV precursor (2.5 mg mL-1) were spin-coated for 60 s at a scan rate of 2000 rpm and converted into linPPV by thermal annealing at 240 °C for 6 h under vacuum.57 (55) Kwon, T. W.; Alam, M. M.; Jenekhe, S. A. Chem. Mater. 2004, 16, 4657– 4666. (56) Stiriba, S. E.; Frey, H.; Haag, R. Angew. Chem., Int. Ed. 2002, 41, 1329– 1334. (57) Schlenoff, J. B.; Wang, L. J. Macromolecules 1991, 24, 6653–6659.

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Article Based on quartz crystal microbalance (QCM) measurements, the thicknesses of the hypPPV and linPPV films were estimated to be 12 and 14 nm, respectively, assuming the film density to be 1.0 g cm-3. For the biopanning experiments, we used thicker films (>80 nm) to avoid peeling or damage, and to avoid exposing the glass substrates during processes such as affinity, washing, and elution procedures. We confirmed the presence of the PPV films throughout the experiments by visually inspecting their greenishyellow colors. Biopanning. The PPV-binding peptides were selected using a commercially available Ph.D.-12 library (New England Biolabs), which provided 2.7  109 different phage clones with 12 amino acid linear-peptide inserts. The phage solution (1.0  1010 plaqueforming units (pfu) in 30 μL of solution) was placed onto the PPV films. After a 5 min incubation at room temperature, the phage solution was removed, and the PPV films were rinsed with Trisbuffered saline (TBS, 50 mM Tris-HCl, 150 mM NaCl, pH 7.5) containing 0.1 wt % Tween-20 once and then TBS four times. The bound phages were eluted from the films by incubating with an elution buffer (100 mM glycine-HCl, 1 mg mL-1 bovine serum albumin, pH 2.2) for 15 min at room temperature, and then the solution was neutralized with 1 M Tris-HCl buffer (pH 9.1). For the next round of biopanning, the eluted phage particles were amplified by infecting with Escherichia coli ER2738 host cells, and then they were purified by poly(ethylene glycol) precipitation. The biopanning process was repeated five times to enrich the phage. After biopanning, the eluted phages were isolated by picking single phage plaques. DNA extracted from 20 clones was sequenced using an Applied Biosystems 3130x1 Genetic Analyzer (Applied Biosystems). Phage Binding Analysis. The binding capabilities of the PPV-binding phages were examined by titer count analysis.36 The corresponding PPV films were prepared in a 96-well glass plate. The films were incubated with a 50 pM phage solution (50 μL/well, 7.5  107 pfu/well) for 1 h at room temperature with gentle shaking. The films were then washed five times with phosphate-buffered saline (PBS, 137 mM NaCl, 8.1 mM Na2HPO4, 2.68 mM KCl, 1.47 mM KH2PO4, pH 7.4) containing 0.1 wt % Tween-20 (200 μL/well). The bound phages were eluted with glycine-HCl (pH 2.2, 50 μL/well) for 10 min at room temperature with gentle shaking. The eluted solutions were collected into new tubes and neutralized with Tris-HCl (pH 9.1, 20 μL/sample), and then PBS (930 μL/sample) was added to give a 1 mL solution. These solutions were further diluted 1:20 with PBS, and then 10 μL aliquots were subjected to titer count assay. SPR Measurements Using Synthetic Peptides. The binding kinetics of the synthetic PPV-binding peptides were examined by using a BIAcore X (GE Healthcare). The peptides were dissolved in HBS-N buffer (10 mM HEPES containing 150 mM NaCl, pH 7.4, GE Healthcare). Freshly prepared peptide solutions were immediately injected into a flow cell at a flow rate of 20 μL min-1 at 25 °C for 3 min. After the injection, the concentrations of the peptide solutions were determined by measuring the absorbance at 280 nm.58 Assuming a Langmuir adsorption model, the association rate constant k1 (M-1 s-1), dissociation rate constant k-1 (s-1), and binding constant Ka (M-1) were estimated. In the case of the Ala-scanning experiments, all peptides were dissolved in HBS-N buffer at a concentration of 50 μM. The association was monitored for 3 min at a flow rate of 20 μL min-1. The amounts bound (ng cm-2) were calculated by increased resonance units (RU) for 3 min from the injection (10 RU = 1 ng cm-2). The obtained values were converted into the bound peptide (pmol cm-2), considering the molecular weight of each peptide. Spectroscopic Methods. The circular dichroism (CD) spectra were recorded on a CD spectropolarimeter (J-725, Jasco). The (58) Pace, C. N.; Vajdos, F.; Fee, L.; Grimsley, G.; Gray, T. Protein Sci. 1995, 4, 2411–2423.

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Figure 2. Percent yield (collected phages/applied phages) against the rounds of biopanning. Table 1. Selected Sequences with Affinity for hypPPV and linPPV clone namea

frequency

pIb

sequence

Hyp01 3/20 7.0 HTDWRLGTWHHS Hyp02 6/20 5.5 NWITMNPAMPTL Hyp03 5/20 5.8 LPFNTLADPRIN Hyp04 5/20 6.9 LLADTTHHRPWT Hyp05 1/20 6.8 NLLIPENVPLRH Lin01 6/20 6.9 ELWSIDTSAHRK Lin02 2/20 8.6 YYPASSTIQSRP Lin03 1/20 9.5 HPTLHMTYYKKQ Lin04 5/20 9.6 HIHRGEHGPSAR Lin05 2/20 9.4 TPLTPNGLTRSG Lin06 1/20 8.8 IVKNVPLTPLRE Lin07 1/20 9.8 AVPHRVGGLHSL Lin08 1/20 6.9 NYLHNHPYGTVG Lin09 1/20 6.0 EHPHVPITPSNL wild type ; ; no displayed peptide a Hyp and Lin mean the target polymers were hypPPV and linPPV, respectively. The numbers represent the order of affinity strength based on the phage binding analysis. b Isoelectric point (pI) calculated using pI/mass program at http://www.expasy.ch.

peptides were dissolved in 10 mM phosphate buffer (pH 7.1) at a concentration of 10 μM. The spectra were recorded in a 1 mm quartz cell over 190-250 nm with buffer baseline subtraction. Ten scans were averaged using a 1 nm bandwidth at a scanning rate of 20 nm min-1. The absorption spectra were recorded on a spectrophotometer (V-550, Jasco). The peptide solution concentrations were determined by their absorbance at 280 nm using molar absorption coefficients calculated from the Trp, Tyr, and Cys content.58

Results and Discussion Selection of the PPV-Binding Phages. To obtain PPVbinding peptides, we performed affinity selections from a phagedisplayed peptide library (Figure 1b). Films of hypPPV and linPPV were prepared on glass substrates. The 12-mer peptide library displayed at the N-terminus of the pIII coat proteins of M13 bacteriophage was applied to the corresponding PPV films. Any unbound phages were removed by washing, and the bound phages were collected by elution. This procedure was repeated five times. In this process, the yield (collected phages/applied phages) showed a continuous increase, and was almost saturated after three rounds possibly due to completion of each selection process (Figure 2). This result indicates that those phages which have a stronger affinity toward the corresponding PPV films were successfully concentrated in the phage pool with proceeding the Langmuir 2010, 26(22), 17278–17285

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biopanning round. Next, the amino acid sequences of the phageexpressed peptides were determined by DNA sequencing. We revealed five and nine different sequences for hypPPV and linPPV, respectively, as listed in Table 1. The frequencies in Table 1 represent the fraction of the same clone from all isolated clones. Multiple appearances of some clones (Hyp01-04, Lin0102, Lin04-05) also suggest that phage libraries were successfully enriched to the objective phage pool in the selection process. Affinities and Specificities of Phage Clones. The amounts of all selected phage clones bound were investigated by phage binding analysis.36 The phage solutions were incubated with PPV films. After washing procedures, the bound phages were collected

Figure 3. Bound amounts of the selected phages against the target films: (a) hypPPV and (b) linPPV. The results are average values with standard deviations from three independent measurements (mean ( SD).

Article

by elution. The phage concentration of the eluted solution was determined by titer count, which quantified the number of eluted phages. We used wild type phage, which displays no peptide, as controls. The bound amounts of phages to the PPV films are shown in Figure 3. On the basis of these results, we assigned names to each PPV-binding clone. For instance, “Hyp04” was denoted that way because the target of the biopanning was hypPPV. The number represents the order of the binding strength; that is, 04 means that this clone had the fourth strongest affinity toward the target. The relative affinities roughly correlated with the frequencies. Most of the clones which had a frequency of 1/20 showed small affinities. All clones, except Lin09, showed superior binding ability as compared to the wild type phage. In particular, Hyp01 and Lin01 showed the strongest affinities. Their relative affinities are 14.2-fold and 9.6-fold greater than that of the wild type phage, respectively. Therefore, we concluded that the displayed 12-mer peptides on the phage bodies apparently enhance their binding ability toward PPV films. Because the Hyp01 and Lin01 sequences have neutral pI values (Table 1), electrostatic interactions do not seem to be a major force for the affinity. To understand the interaction between selected sequences and PPV, the percent appearances of amino acids in the obtained sequences (taking the frequency into account) were calculated and are shown in Figure 4. Since the percentage of each amino acid in the original random library is known, we can investigate which amino acids were enriched during the biopanning process by comparing the percent values with that of the original library. The percentages of Trp, His, Arg, Ile, and Ala were increased in both cases (hypPPV and linPPV). Trp and His have aromatic side chains; thus, π-π interactions between PPV’s phenylene rings and the side chains of Trp and His might play a decisive role. This speculation is further confirmed by an Ala-substitution study (see below). Ile and Ala enrichment is possibly due to hydrophobic interactions or CH-π interactions. His and Arg might be able to participate in the interaction through cation-π interactions.59 Additionally, since Arg and Ile have relatively large side chains, these amino acids can regulate the conformation of the peptides. Interestingly, the enrichment of Trp and His has also been reported in CNT-binding peptides.29,31,32 More interestingly, the Hyp04 sequence has already been reported as a CNT-binding peptide.31 This is reasonable because both PPV and CNT are composed of conjugated CdC double bonds. The clone was selected from the phage-displayed peptide library in both cases,

Figure 4. Percentage of amino acids in the obtained peptide sequences. Langmuir 2010, 26(22), 17278–17285

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probably due to their high affinity toward the benzene rings or conjugated carbons. On the other hand, to the best of our knowledge, the Hyp01 and Lin01 sequences were isolated for the first time from this library. This suggests that Hyp01 and Lin01 have not only high affinity but also high specificity toward the corresponding PPV. Among the selected phage clones listed in Table 1, we focused on Hyp01, Lin01, and Hyp04 for further investigations. This is because Hyp01 and Lin01 showed the strongest affinity for the

Figure 5. Characterization of PPV-binding sequences. (a) Amino acid sequences of Hyp01, Lin01, and Hyp04. The sequence homologies are highlighted by enclosing them in boxes. Red, aromatic; green, hydrophobic; blue, basic; and purple, hydrophilic amino acids. (b) Binding specificities of Hyp01, Lin01, Hyp04, and wild type phage clones against hypPPV or linPPV films and bare glass substrates. The results are average values with standard deviations from three independent measurements (mean ( SD).

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corresponding PPV films. Additionally, although Hyp04 showed only moderate affinity toward hypPPV, that is, 2.5-fold greater than that of the wild type phage, Hyp04 was also investigated because this sequence is already known as a CNT-binding peptide.31 Moreover, a consensus sequence was found among these three sequences (Trp-Xaa-Leu/Ile/Ala-Asp-Thr-Trp/Ser/ThrHis-His-Arg), as shown in Figure 5a. This motif contains four aromatic amino acids (His and Trp), located at specific positions separated by amphiphilic spacers. As mentioned in Figure 4, Trp and His are considered to be important amino acids due to their enrichment among the selected sequences. The amphiphilic spacers probably fix the location of these aromatic amino acids, allowing the peptides to interact with the PPV by π-π interactions at the appropriate positions (see below). Next, we examined the specificity of the Hyp01, Lin01, and Hyp04 clones for hypPPV and linPPV. The phage solution was incubated with the PPV films, and the bound phages were quantified by titer count analysis, as shown in Figure 5b. All clones showed low amounts bound on bare glass substrates. In contrast, these clones showed much larger affinities for PPV films. Additionally, Hyp01 showed a 2.1-fold greater binding ability for hypPPV rather than linPPV, whereas Lin01 showed a 9.8-fold greater binding ability toward linPPV rather than hypPPV. On the other hand, the bound amount of Hyp04 to hypPPV was obviously smaller than that of Hyp01 or Lin01 and Hyp04 did not show any such specificity. Although Hyp04 was concentrated from a phage library, the binding ability of the clone seemed to be less than that of the other two clones. Furthermore, the wild type phage did not show significant binding and specificity. hypPPV and linPPV are both composed of benzene rings and CdC double bonds, but hypPPV is branched and linked through the meta-positions, and linPPV is linearly connected through the

Figure 6. SPR experiments. (a-d) SPR sensorgrams of (a) Hyp01 peptide for hypPPV, (b) Hyp01 peptide for linPPV, (c) Lin01 peptide for hypPPV, and (d) Lin01 peptide for linPPV at various concentrations. (e,f) Plots of the observed rate constant (kobs) against the concentration of the peptides. 17282 DOI: 10.1021/la102018f

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Table 2. SPR Kinetic Parameters, Association Rate (k1), Dissociation Rate (k-1), and Binding Constants (Ka) of the PPV-Binding Peptides peptide

polymer

k1 (102 M-1 s-1)

k-1 (10-3 s-1)

Ka (104 M-1)

Hyp01

hypPPV linPPV hypPPV linPPV

16 4.0 1.2 2.1

2.1 7.6 3.3 2.7

77 5.2 3.6 7.7

Lin01

para-positions (Figure 1a). The results clearly show that the Hyp01 and Lin01 clones have potentials to differentiate between such small structural differences. Affinities and Specificities of Chemically Synthesized Peptides. In the phage binding analysis, we quantified the number of phage particles bound to the polymer film surfaces. Since whole phage particles (ca. 1 μm long and 6 nm in diameter)60 are much larger than the displayed 12-mer peptides, the results of the phage binding analysis did not provide direct evidence of the peptide binding affinity. Therefore, we selected the strongest binding sequences, Hyp01 and Lin01, for the binding assay in synthetic peptide level. The SPR measurements were performed in order to quantitatively assess the binding constants for the synthetic Hyp01 and Lin01 peptides, which are free from the phage particles, as shown in Figure 6. The bound amounts of the peptides were successfully determined with affinity time, and were increased with increasing the peptide concentration. Assuming a Langmuir adsorption model,21 the data were fitted to obtain the association (k1)/dissociation (k1) rate constants and the binding constant (Ka) from their ratio. The resulting parameters are summarized in Table 2. The combination which showed the highest Ka value was Hyp01-hypPPV, and reached 7.7  105 M-1. This value is comparable to or slightly larger than our previous report of a 7-mer it-PMMA-binding peptide (c02-itPMMA, Ka = 2.8  105 M-1).40 Although the Ka is sufficiently large for the short peptide affinity, it is probably possible to enhance this value by preparing tandem multiple repeats,24 cyclization,61 or displaying on a dendrimer.62 With respect to specificity, the Hyp01 peptide showed a 15-fold greater binding constant toward hypPPV rather than linPPV (7.7  105 M-1 vs 5.2  104 M-1). The Lin01 peptide also showed a 2.1-fold greater binding constant for linPPV rather than hypPPV (7.7  104 M-1 vs 3.6  104 M-1). Thus, the specificities of the PPV-binding peptides, Hyp01 and Lin01, were completely maintained, even if the peptides were free from phage particles. Atomic force microscopic (AFM) observations showed that films of hypPPV and linPPV have a similar roughness and surface area (Figure S1 in the Supporting Information). Therefore, it is evident that the specificity of the synthetic peptides, Hyp01 and Lin01, is simply derived from their molecular recognition properties. Ala-Scanning of the Hyp01 Peptide. To understand more about the binding mechanisms at the molecular level, we investigated the Hyp01 peptide in more detail and synthesized a series of the Hyp01 variants in which amino acids are substituted by Ala residues (side chain R = CH3) one by one (so-called, Alascanning). Although Gly (R = H) also nullifies the side chain and could introduce conformational flexibility into the backbone,63 we chose Ala for scanning, because the Hyp01 peptide (59) Gallivan, J. P.; Dougherty, D. A. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 9459–9464. (60) Smith, G. P.; Petrenko, V. A. Chem. Rev. 1997, 97, 391–410. (61) Seker, U. O. S.; Wilson, B.; Dincer, S.; Kim, I. W.; Oren, E. E.; Evans, J. S.; Tamerler, C.; Sarikaya, M. Langmuir 2007, 23, 7895–7900. (62) Helms, B. A.; Reulen, S. W. A.; Nijhuis, S.; de Graaf-Heuvelmans, P.; Merkx, M.; Meijer, E. W. J. Am. Chem. Soc. 2009, 131, 11683–11685. (63) Morrison, K. L.; Weiss, G. A. Curr. Opin. Chem. Biol. 2001, 5, 302–307.

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Figure 7. Results of the Ala-scanning experiments. The amounts bound of each Ala mutant of the Hyp01 peptide were determined by SPR. The results are average values with standard deviations from three independent measurements (mean ( SD).

Figure 8. Amounts bound of the Hyp01, W4A, and W9A peptides on hypPPV and linPPV films, as determined by SPR. The results are average values with standard deviations from three independent measurements (mean ( SD).

Figure 9. CD spectra of the Hyp01, Lin01, and Hyp04 peptides.

originally contains no Ala but Gly. Additionally, the power of Ala-scanning to provide critical insight of binding mechanisms has already been demonstrated.16,40 The bound amounts of the synthesized 12 Ala mutants were determined by SPR (Figure 7). DOI: 10.1021/la102018f

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Figure 10. Binding mechanism of the Hyp01 peptide. (a) Possible conformation of the Hyp01 peptide. Hydrogen atoms were omitted for clarity. Blue, nitrogen; red, oxygen; gray, carbon. (b) 3D molecular docking of Hyp01-hypPPV. The distance between the two Trp residues fits that of the intramolecular benzene rings three repeating units away in hypPPV. For clarity, only the two Trp side chains are shown. (c) 3D molecular model of linPPV. The distance (1.4 nm) does not fit with that of linPPV’s intramolecular benzene rings (0.6, 1.2, and 1.8 nm).

A substitution at any position of the Hyp01 peptide by Ala markedly decreased the affinity, suggesting that all amino acids are essential for the strong interaction. However, the degree of decrease differed from sequence to sequence. We determined the order of importance from the decreased magnitude of the amounts bound as follows: Trp9 > Trp4 > Arg5 > Leu6 > Gly7 > His10 > His1 > Thr8 > Thr2 > Asp3 > His11 > Ser12. It was therefore revealed that the two Trp residues which were expected to form π-π interactions were key residues for this interaction. Interestingly, the following important amino acids Arg5, Leu6, and Gly7 are located between the two Trp residues. Assuming that Arg participates in cation-π interactions and Leu and Gly participate in hydrophobic interactions as mentioned above, one can speculate that the amphiphilic spacer consisting of these three residues fixes the conformation of the peptide to locate the two Trp residues at the appropriate positions. Next, the His10 and His1 follow. Thus, the π-π interactions of Trp rather than His are more important for the interaction between the peptide and PPV. Similar results have been obtained for CNT-binding peptides.33 Interestingly, the important region of the Hyp01 sequence determined from Ala-scanning experiments, Trp4-His10, gave close agreement with the consensus sequence (Figure 5a). It should be noted that the three sequences shared homology but the most important amino acid, Trp9, existed only in the Hyp01 sequence, thereby indicating that the Trp9 residue plays a special role in the specificity for hypPPV. Plausible Binding Mechanism of the Hyp01 Peptide. The underlying mechanism responsible for the specificity of PPVbinding peptides toward structurally similar PPV targets is not fully understood at present, but we propose a plausible binding mechanism for the Hyp01 peptide, as follows. By the Ala substitution of Trp (W4A and W9A), not only the binding ability but also the specificity were dramatically reduced (Figure 8). Since only single Ala substitution can drastically change the specificity of Hyp01, two Trp residues work cooperatively for the interaction. To study the conformation of peptides, we measured CD spectra of the Hyp01, Lin01, and Hyp04 peptides (Figure 9). The Lin01 and Hyp04 peptides had spectra typical of a random coil. On the other hand, in the case of the Hyp01 peptide, a positive cotton effect was observed at around 228 nm, suggesting that the peptide partially adopted a polyproline type II (PII) helical conformation (left-handed 31 helix).22,64 This PII conformation was completely deformed to a random coil conformation by the substitutions of Trp to Ala (Figure S2a in the Supporting (64) Chellgren, B. W.; Creamer, T. P. Biochemistry 2004, 43, 5864–5869.

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Information), suggesting that the conformational state strongly affects its binding affinity. In fact, the decrease in peak intensity assigned to the PII conformation was remarkable only by the Trp substitutions, suggesting the significance of the two Trp residues for the conformational regulation (Figure S2b in the Supporting Information). In other words, even if the Hyp01 variants could form the PII conformation, the absence of amino acid residues in the possible motif sequence (Trp-Arg-Leu-Gly-Thr-Trp-His) of the Hyp01 peptide resulted in obvious decreases in binding affinities (see Figure 7), also supporting the possibility that not ony Trp residues but also other amino acids in the motif cooperatively participate in the binding to hypPPV, as aforementioned. We constructed a possible 3D structure of the Hyp01 peptide (Figure 10a), assuming the peptide formed the PII conformation even on the surface of hypPPV. The initial structure was set to the conformation, and the obtained structure was further optimized by molecular mechanics calculations. In this model, the two Trp residues were located on one side of the helix, and the distance between them was estimated to be 1.4 nm, which corresponds to that of the benzene rings three repeating units away in hypPPV (Figure 10b). On the other hand, the distance does not fit those of the intramolecular benzene rings in linPPV (Figure 10c). Although there are some other amino acids in Hyp01 that are likely to participate in this interaction, we believe that the specificity of the Hyp01 peptide was due principally to the aforementioned two Trp residues, because the substitution of one Trp residue resulted in the loss of this specificity. In a similar fashion, the 3D structure of the Lin01 peptide was also constructed (Figure S3 in the Supporting Information). Although the Ala-scanning experiments on the Lin01 peptide have still not been conducted, there should be another amino acid responsible for the specificity, because this sequence contains only one Trp residue. In this case, the His is a plausible candidate because the distance between the Trp and His residues is 2.4 nm, which fits the 4 repeating units in linPPV.

Conclusions We have identified PPV-binding peptides by the phage display method. The aromatic amino acids were enriched in these sequences, suggesting that π-π interactions between the peptides and PPV were essential. The affinity constant of the Hyp01 peptide for hypPPV was estimated to be 7.7  105 M-1, which was 15-fold greater than that obtained for linPPV. Thus, we clearly demonstrated that the screened peptides could recognize the branched or linear structure of PPV. A sequence motif for the interaction between the Hyp01 peptide and hypPPV, TrpArg-Leu-Gly-Thr-Trp-His, was successfully proposed due to the Langmuir 2010, 26(22), 17278–17285

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Ala-scanning experiments. In fact, this motif was nearly identical with the consensus sequence found between the Hyp01, Lin01, and Hyp04 peptides. Slight deviations from this consensus sequence resulted in different affinities and specificities. We discussed a plausible molecular binding model explaining the specificity of the Hyp01 peptide. The PII helical conformation allowed the peptide to locate its two Trp residues at appropriate face positions. These Trp residues (and other residues might also play a role) may fit into hypPPV and interact in a multivalent fashion. Applications such as solubilization of hypPPV in aqueous phases and developing novel bioimaging or biosensing systems using PPV-binding peptides are currently in progress, and will be reported elsewhere in the near future.

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Acknowledgment. We thank Prof. M. Komiyama (the University of Tokyo) for MALDI-TOF MS and CD spectral measurements and Prof. H. Aburatani (the University of Tokyo) for DNA sequence analysis. This work was supported in part by Global COE Program (Chemistry Innovation through Cooperation of Science and Engineering) and Grant-in-Aid for Scientific Research (Nos. 20350052 and 21106506). H.E. also thanks Grant-in-Aid for JSPS Fellows (No. 21-4887). Supporting Information Available: Synthesis of peptides, AFM images, CD spectra of Ala mutants, and possible conformation of the Lin01 peptide. This material is available free of charge via the Internet at http://pubs.acs.org.

DOI: 10.1021/la102018f

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