DOI: 10.1021/la901242q 10901 Langmuir 2009, 25(18), 10901–10906 Published on Web 07/16/2009 pubs.acs.org/Langmuir © 2009 American Chemical Society Critical Amino Acid Residues for the Specific Binding of the Ti-Recognizing Recombinant Ferritin with Oxide Surfaces of Titanium and Silicon Tomohiro Hayashi,* ,†,‡ Ken-Ichi Sano, §,z Kiyotaka Shiba, § Kenji Iwahori, ^ Ichiro Yamashita, ^, ) and Masahiko Hara †,‡ † Department of Electronic Chemistry, Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8502, Japan, ‡ Flucto- Order Functions Asian Collaboration Team, Advanced Science Institute, RIKEN, 2-1 Hirosawa, Wako, Sait- ama 351-0198, Japan, § Department of Protein Engineering, Cancer Institute, Japanese Foundation for Cancer Research, 3-10-6 Ariake, Koto-ku, Tokyo 135-8550, Japan, and CREST, JST, 4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8502, Japan, ^ Graduate School of Materials Science, Nara Institute of Science and Technology, and CREST, JST, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan, and ) Advanced Technology Research Laboratories, Matsushita Electric Industrial Co., Ltd., 3-4 Hikari-dai, Seika, Kyoto 619-0237, Japan. z Present address: Advanced Science Institute, RIKEN Received April 8, 2009. Revised Manuscript Received June 20, 2009 The interactions of ferritins fused with a Ti-recognizing peptide (RKLPDA) and their mutants with titanium oxide substrates were explored with an atomic force microscope (AFM). The amino acid sequence of the peptide was systematically modified to elucidate the role of each amino acid residue in the specific interaction. Force measurements revealed a clear correlation among the sequences in the N-terminal domain of ferritin, surface potentials, and long-range electrostatic interactions. Measurements of adhesion forces clearly revealed that hydrogen bonds take part in the specific binding as well as the electrostatic interaction between charged residues and surface charges of Ti oxides. Moreover, our results indicated that not only the charged and polar residues but also a neutral residue (proline) govern the strength of the specific binding, with the order of the residues also being significant. These results demonstrate that the local structure of the peptide governs the special arrangement of charged residues and strongly affects the strength of the bindings. Introduction Peptide aptamers (binders) with specific affinity to their target materials have been opening up new avenues for the construction of hybrid bioinorganic interfaces. 1,2 So far, se- quences exhibiting specificity to metals, 3-6 semiconducting materials, 7-13 and other organic nanocompounds such as nanocarbon materials 14 have been reported. By using their selectivity, specificity and capability of biomineralization, formations of bioinorganic interfaces with simple and easy processes have been demonstrated elsewhere. 15-18 The sequences of target-specific peptide have been acquired by biopanning processes with phage or cell surface display techni- ques. 1,2 With these approaches, many short peptides specifically binding to metals, oxides, and semiconductors have been re- ported. 2,6 Recently, Peelle et al. and Willett et al. attempted to design peptides based on databases on the adhesion coefficient of each animo acid to various materials. 19,20 Oren et al. analyzed the sequence similarity of peptides specifically binding to quartz and demonstrated the knowledge-based design of peptides bound to quartz. 21 These works will lead to predictive design of peptide aptamers targeting various materials. In spite of the above successes in the designs of target-specific peptides, the understanding of the specific binding between peptides and target materials at a microscopic level has been rather limited because of the lack of information about the physical origin of the binding. Previous studies confirmed that charged polar residues (Lys, Arg, His, Asp, and Glu) expressed strong affinity to materials that are covered with a natural oxide layer, implying that electrostatic interactions, such as interaction between charges and hydrogen bond-type interactions, signifi- cantly take part in the specific binding. On the other hand, the reported sequences of the peptides frequently contain non- polar residues such as proline residues, clearly suggesting the importance of the structures of the peptides. 4,7,20 To understand the mechanism underlying the specific binding between peptides and their targets, a quantitative analytical method to characterize the interaction is necessary. *Corresponding author e-mail: hayashi@echem.titech.ac.jp. (1) Baneyx, F.; Schwartz, D. T. Curr. Opin. Biotechnol. 2007, 18, 312. (2) Tamerler, C.; Sarikaya, M. Acta Biomater. 2007, 3, 289. (3) Brown, S. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 8651. (4) Sano, K.; Shiba, K. J. Am. Chem. Soc. 2003, 125, 14234. (5) Naik, R. R.; Brott, L. L.; Clarson, S. J.; Stone, M. O. J. Nanosci. Nanotechnol. 2002, 2, 95. (6) Sarikaya, M.; Tamerler, C.; Jen, A. K. Y.; Schulten, K.; Baneyx, F. Nat. Mater. 2003, 2, 577. (7) Whaley, S. R.; English, D. S.; Hu, E. L.; Barbara, P. F.; Belcher, A. M. Nature 2000, 405, 665. (8) Schembri, M. 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I.; Yoshii, S.; Yamashita, I.; Shiba, K. Nano Lett. 2007, 7, 3200. (19) Peelle, B. R.; Krauland, E. M.; Wittrup, K. D.; Belcher, A. M. Langmuir 2005, 21, 6929. (20) Willett, R. L.; Baldwin, K. W.; West, K. W.; Pfeiffer, L. N. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 7817. (21) Oren, E. E.; Tamerler, C.; Sahin, D.; Hnilova, M.; Seker, U. O. S.; Sarikaya, M.; Samudrala, R. Bioinformatics 2007, 23, 2816. Downloaded by TOKYO INST OF TECH on September 13, 2009 | http://pubs.acs.org Publication Date (Web): July 16, 2009 | doi: 10.1021/la901242q