264
Bioconjugate Chem. 2010, 21, 264–269
Modification of Porous Protein Crystals in Development of Biohybrid Materials Tomomi Koshiyama,† Naomi Kawaba,§ Tatsuo Hikage,‡ Masanobu Shirai,† Yuki Miura,§ Cheng-Yuan Huang,| Koichiro Tanaka,† Yoshihito Watanabe,| and Takafumi Ueno*,†,⊥ Institute for Integrated Cell-Material Sciences (iCeMS), Funai Center, Kyoto University Katsura, Nishikyo-ku, Kyoto 615-8510, Japan, Department of Chemistry, Graduate School of Science, High Intensity X-ray Diffraction Laboratory, and Research Center for Materials Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan, PRESTO, Japan Science and Technology Agency (JST), Honcho Kawaguchi, Saitama 332-0012, Japan. Received July 11, 2009; Revised Manuscript Received December 22, 2009
Protein assemblies have attracted increasing attention for construction of biohybrid materials. Protein crystals can also be regarded as solid protein assemblies. The present work demonstrates that protein crystals can be employed as porous biomaterials by site-specific modifications of the crystals of recombinant sperm whale myoglobin mutants. The myoglobin crystals of space group P6 provide hexagonal pores consisting of the building blocks of six Mb molecules, which form a pore with a diameter of 40 Å. On the basis of the lattice structure of the Mb crystals, we have selected appropriate residues located on the surface of the pores for replacement with cysteine. This enables modification of the pore surface via coupling with maleimide derivatives. We have succeeded in crystallizing the modified Mb mutants, retaining the P6 lattice, and consistently aligning nanosized functional molecules such as fluorescein, eosin, and Ru(bpy)3 into the hexagonal pores of the Mb crystals. Our strategy for site-specific modification of protein crystal pores is applicable to various protein crystals with porous structures. We believe that modified porous protein crystals will provide attractive candidates for novel solid materials in nanotechnology applications.
INTRODUCTION It has been recently recognized that the design of protein assemblies holds promise for construction of biohybrid materials which have the potential to play crucial roles in the field of biotechnology (1–8). Efforts to modify nanosized space of caged, tubular, and two-dimensional protein assemblies have recently attracted increasing attention because nanospaces are expected to provide sufficient space to accommodate large and complex functional molecules for biotechnological applications (3, 6, 8–13). Protein crystals can also be regarded as solid protein assemblies. Malgolin et al. have indicated that protein crystals consist of highly ordered protein assemblies with unique nanosized porous structures (14, 15). The protein crystals comprise solvent-filled pores ranging from 30% to 65% of the total crystal volume with the advantageous properties of high porosity (0.5-0.8), large surface area (800-2000 m2/g), and a wide range of pore sizes (20-100 Å). These properties comparable to typical porous materials suggest that pores of protein crystals are able to provide enough spaces to promote various reactions (15–17). Protein crystals are able to serve as heterogeneous catalysts even under extreme conditions including high temperature, organic solvents, and extreme acidity or basicity when stabilized by cross-linkages (14). A recent report has described that soaking of cross-linked virus crystals in a buffer solution * Institute for Integrated Cell-Material Sciences (iCeMS), Funai Center, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan. Tel/Fax: +81-75-383-2812. E-mail:
[email protected]. † iCeMS, Kyoto University. § Department of Chemistry, Graduate School of Science, Nagoya University. ‡ High Intensity X-ray Diffraction Laboratory, Nagoya University. | Research Center for Materials Science, Nagoya University. ⊥ PRESTO, Japan Science and Technology Agency (JST).
containing metal ions causes the metal ions to be deposited within the porous spaces of the crystals and converted into metallic materials in the presence of reducing reagents (18). It has been recognized that transport of water, ions, and organic molecules in protein channels are crucial processes in various biological reactions. Experimental and theoretical studies of diffusion and absorption of small molecules in porous protein crystals have been reported (19, 20). These reports suggest that porous protein crystals may be useful as biohybrid materials if the pore surfaces could be selectively modified by a combination of site-directed mutagenesis and bioconjugation techniques. However, there have been no reports of modification of the pore surfaces of porous protein crystals because the consensus has been that even small a amount of modified proteins (
