Toru Tsuji

Direct transformation from amorphous to crystalline calcium phosphate facilitated by motif-programmed artificial proteins

An animals hard tissue is mainly composed of crystalline calcium phosphate. In vitro, small changes in the reaction conditions affect the species of calcium phosphate formed, whereas, in vivo, distinct types of crystalline calcium phosphate are formed in a well-controlled spatiotemporal-dependent manner. A variety of proteins are involved in hard-tissue formation; however, the mechanisms by which they regulate crystal growth are not yet fully understood. Clarification of these mechanisms will not only lead to the development of new therapeutic regimens but will also provide guidance for the application of biomineralization in bionanotechnology. Here, we focused on the peptide motifs present in dentin matrix protein 1 (DMP1), which was previously shown to enhance hydroxylapatite (HAP) formation when immobilized on a glass substrate. We synthesized a set of artificial proteins composed of combinatorial arrangements of these motifs and successfully obtained clones that accelerated formation of HAP without immobilization. Time-resolved static light-scattering analyses revealed that, in the presence of the protein, amorphous calcium phosphate (ACP) particles increased their fractal dimension and molecular mass without increasing their gyration radii during a short period before precipitation. The protein thus facilitated reorganization of the internal structure of amorphous particles into ordered crystalline states, i.e., the direct transformation of ACP to HAP, thereby acting as a nucleus for precipitation of crystalline calcium phosphate. Without the protein, the fractal dimension, molecular mass, and gyration radii of ACP particles increased concurrently, indicating heterogeneous growth transformation.

Directional BMP-2 for functionalization of titanium surfaces.

Efficient immobilization of biomacromolecules on material surfaces is a key to development in areas of regenerative medicine and tissue engineering. However, strong and irreversible immobilization of cytokines on surfaces often diminishes their biological functionality. A destructive hydrophobic interaction between the material surface and the biomolecule may underlie this inactivation. Alternatively, dissociation of the cytokine from the material may be necessary for signal transduction. Here we propose a new method for immobilizing cytokines on material surfaces: a material-binding artificial peptide is used to mediate reversible interaction between the cytokine and the material surface. We created artificial proteins that contained three copies of a Ti-binding motif, and fused them to the N-terminal of BMP-2. The engineered BMP-2 showed reversible binding to Ti surfaces and induced BMP signaling activity. When a hydrophobic protein devoid of the Ti-binding motif was fused to BMP-2, the protein tightly bound to Ti surfaces but showed little BMP activity, confirming the importance of the mode of immobilization.

mRNA display selection of a high-affinity, Bcl-XL-specific binding peptide

Bcl‐XL, an antiapoptotic member of the Bcl‐2 family, is a mitochondrial protein that inhibits activation of Bax and Bak, which commit the cell to apoptosis, and it therefore represents a potential target for drug discovery. Peptides have potential as therapeutic molecules because they can be designed to engage a larger portion of the target protein with higher specificity. In the present study, we selected 16‐mer peptides that interact with Bcl‐XL from random and degenerate peptide libraries using mRNA display. The selected peptides have sequence similarity with the Bcl‐2 family BH3 domains, and one of them has higher affinity (IC50=0.9 µM) than Bak BH3 (IC50=11.8 µM) for Bcl‐XL in vitro. We also found that GFP fusions of the selected peptides specifically interact with Bcl‐XL, localize in mitochondria, and induce cell death. Further, a chimeric molecule, in which the BH3 domain of Bak protein was replaced with a selected peptide, retained the ability to bind specifically to Bcl‐XL. These results demonstrate that this selected peptide specifically antagonizes the function of Bcl‐XL and overcomes the effects of Bcl‐XL in intact cells. We suggest that mRNA display is a powerful technique to identify peptide inhibitors with high affinity and specificity for diseaserelated proteins.—Matsumura, N., Tsuji, T., Sumida, T., Kokubo, M., Onimaru, M., Doi, N., Takashima, H., Miyamoto‐Sato, E., Yanagawa, H. mRNA display selection of a high‐affinity, Bcl‐XL‐specific binding peptide. FASEBJ. 24, 2201–2210 (2010). www.fasebj.org

Towards the Creation of Novel Proteins by Block Shuffling

We have been investigating the creation of novel proteins by means of block shuffling, where the term block refers to an amino acid sequence that corresponds to particular features of proteins, such as secondary structures, modules, functional motifs, and so on. Block shuffling makes it possible to explore the global sequence space, which is not feasible with conventional methods, such as DNA shuffling or family shuffling. To investigate what properties are required for the building blocks, we have analyzed the foldability and enzymatic activity of barnase mutants obtained by permutation of modules or secondary structure units. This reconstructive approach indicated that secondary structure units with mutual long-range interactions are more suitable than modules as building blocks, at least in the case of barnase. The results also suggested that proteins in evolutionarily intermediate states are created by block shuffling, and such proteins have the potential to be evolved into mature globular proteins. For the construction of combinatorial protein libraries, we have developed random multi-recombinant PCR (RM-PCR), which can combine different DNA fragments without homologous sequences. The libraries can be utilized for in vitro selection using in vitro virus (mRNA display) or stable (DNA display), which have also been developed in our laboratory. In this review article, we summarize our strategy to create novel proteins by block shuffling and review key literature in the field. Possible applications of the block shuffling strategy are also discussed.

In vitro selection of GTP-binding proteins by block shuffling of estrogen-receptor fragments

To what extent has alternative splicing contributed to the evolution of protein-function diversity? We previously constructed a pool of block-deletion mutants of the human estrogen receptor alpha ligand binding domain by random multi-recombinant PCR. Here we performed iterative in vitro selection of GTP-binding proteins by using the library of mRNA-displayed proteins and GTP-affinity chromatography combined with quantitative real-time PCR. We obtained a novel GTP-binding protein with moderate affinity and substrate-specificity. The results of our in vitro simulation imply that alternative splicing may have contributed substantially to the diversification of protein function during evolution.

Random multirecombinant polymerase chain reaction.

Publisher Summary This chapter examines the random multirecombinant polymerase chain reaction (PCR). Random multirecombinant (RM) PCR that permits the shuffling of several DNA fragments without homologous sequences is developed. RM-PCR is based on multirecombinant PCR, which is a modified method of overlap extension PCR. In multirecombinant PCR, several dimer templates having overlapped segments are combined in a single PCR. It is found that through the use of dimer templates encoding two peptide sequences, a structural gene consisting of several building blocks is created. It is suggested to create different structural genes simultaneously, different dimer templates are mixed such that at least one segment of a dimer template can overlap with more than two different dimer templates. Dimer templates may be obtained by chemical synthesis as single-stranded DNAs if the building blocks are short sequences. These single-stranded DNAs can be converted to double-stranded DNAs by PCR or hybridization. It is found that most of the block sequences in the combinatorial libraries created by RM-PCR encode a long open reading frame and are suitable for protein selection experiments.

Artificial peptides binding to the c face of hydroxyapatite obtained by molecular display technology

Artificial liner peptides that bind to the c face of hydroxyapatite (HA) were obtained by using the mRNA display method. The specific attachment to the c face was characterized with the adsorption isotherm of the peptides. The crystal growth of HA was found to be modulated by the particular peptides. The results of this study suggest that the amino acid sequence including Ala-Asn-Thr (ANT) is essential for the binding specificity.

N- and C-terminal fragments of a globular protein constructed by elongation of modules as a units associated for functional complementation.

We have been interested in partially folded proteins with marginal stability and activity, because they have a potential to be mature proteins by artificial evolution. A module is defined as a contiguous peptide chain forming a compact region in a globular protein. Modules may be used as building blocks to create partially folded proteins. Barnase, a ribonuclease consisting of 110 amino acids, has been divided into six modules (M1-M6), four peptide fragments, M12 (1-52), M123 (1-73), M1234 (1-88) and M12345 (1-98), have been constructed by progressive elongation of the modules from the N-terminus. Only M12345 (1-98) had a partially folded conformation, but it lacked detectable RNase activity. A mixture of M12345 (1-98) with M56 (89-110) showed weak but distinct RNase activity. Unfolded M12345 (1-96) was constructed by removal of two residues from the C-terminus of M12345 (1-98). The mixture of M12345 (1-96) with M56 (89-110) also showed RNase activity. Further, the interaction endowed M12345 (1-96) with conformational stability. We propose that N- and C-terminal fragments obtained by successive elongation of modules would interact to be a complex with marginal stability and activity, which would be used for creating a mature complex by artificial evolution.

Biomineralization: Tooth Enamel Formation

Tooth enamel is composed of well-crystallized apatite that elongates in the c-axis direction with a highly organized orientation. How do these crystals form? It is still an unanswered question, despite enormous efforts made on answering it.

Biomineralization: Apatite Protein Interaction

The evolution of recombinant DNA techniques and protein engineering has accelerated the growth in biomineralization studies over the last decade. In this chapter, we discuss recently published work focusing on the structure and function of proteins that are involved in HAP crystal formation in the body. The proteins we focus on in this review are amelogenin and dentin matrix protein 1 (DMP1). The roles of other proteins, for example, SIBLING family members, which are supposed to play significant roles in HAP crystal formation, are also described. These proteins would be involved in different steps of HAP crystal formation, that is, nucleation, growth, and transformation. We also summarize the challenges of regulating crystal growth and elucidating the mechanisms of crystal formation using artificial proteins, which are not attained by using only naturally occurring proteins.