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

Abstract

An animal's 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 wellcontrolled 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. biomaterials ͉ biomineralization ͉ crystal growth ͉ protein engineering H ydroxylapatite (Ca 10 (PO 4 ) 6 (OH) 2 , HAP) is a major component of bone and teeth (1). Formation of HAP in vitro is readily affected by the small changes in reaction conditions (2-4), whereas, in vivo, it is formed under the robust biomineralization process. Various proteins have been proposed to be involved in the biomineralization of HAP (5-7). Dentin matrix protein1 (DMP1) is one of such biomineralization proteins, and the ablation of its gene results in bone malformation (8-11). He et al. (6) have shown that two peptide motifs identified in DMP1 [motif-A (ESQES) and motif-B (QESQSEQDS)] enhanced in vitro HAP formation when immobilized on a glass plate (6). Along with DMP1's motifs, several peptides and proteins have been shown to act as accelerators of HAP formation, and it has been suggested that these molecules mediate nucleation during crystal formation (12)(13)(14). The purpose of our experiment was to gain insight into the molecular mechanism by which the peptide motifs of DMP1 facilitate HAP formation through our synthetic approach (15). Because immobilization of a specimen poses impediments to time-resolved analyses, such as lightscattering photometry (16, 17), we first synthesized motifprogrammed artificial proteins from the t...

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