Amelogenins make up a class of proteins associated with the formation of mineralized enamel in vertebrates, possess highly conserved N-and C-terminal sequence regions, and represent an interesting model protein system for understanding biomineralization and protein assembly. Using bioinformatics, we report here the identification of molecular traits that classify 12 amelogenin proteins as members of the intrinsically disordered or unstructured protein family (IDPs), a group of proteins that normally exist as unfolded species but are capable of transformation to a folded state as part of their overall function. Using biophysical techniques (CD and NMR), we follow up on our bioinformatics studies and confirm that one of the amelogenins, recombinant porcine rP172, exists in an extended, unfolded state in the monomeric form. This protein exhibits evidence of conformational exchange between two states, and this exchange may be mediated by Pro residues in the sequence. Although the protein is globally unfolded, we detect the presence of local residual secondary structure [α-helix, extended β-strand, turn/loop, and polyproline type II (PPII)] that may serve several functional roles within the enamel matrix. The extended, labile conformation of rP172 amelogenin is compatible with the known functions of amelogenin in enamel biomineralization, i.e., self-assembly, associations with other enamel matrix proteins and with calcium phosphate biominerals, and interaction with cell receptors. It is likely that the labile structure of this protein facilitates interactions of amelogenin with other macromolecules or with minerals for achievement of internal protein stabilization.The formation of inorganic compounds by organisms (biomineralization) is a substantial scientific puzzle. The ability of cells to employ proteins to control nucleation, crystal morphology, polymorphism, and the material properties of living tissues requires precise molecular control and efficient mechanisms (1). One such protein, amelogenin, is found in mammalian tooth enamel, one of the most highly mineralized materials of vertebrates (1-3). Amelogenin is essential for normal enamel development and is capable of protein selfassociation, forming supramolecular assemblies under defined conditions in the laboratory (4-6). These supramolecular assemblies (nanospheres) are believed to exert control over the † This work was supported by funding from the Department of Energy (DE-FG02-03ER46099) and the National Institute of Dental and Craniofacial Research (DE-013414 morphology, organization, and directionality of hydroxyapatite crystal growth (7,8). Primary sequence analysis of 26 mammalian lineages indicates that the N-terminus (Tyr-rich) and Cterminus (charged) of amelogenin are highly conserved, whereas variations occur in the central regions (9). Amelogenin sequence mutations lead to defective enamel crystal formation and organization (10,11), and deletion of the conserved terminal domains leads to the formation of ill-defined enamel crystals, high...

Reconstructing enamel-like structures on teeth has been an important topic of study in the material sciences and dentistry. The important role of amelogenin in modulating the mineralization of organized calcium phosphate crystals has been previously reported. We used amelogenin and utilized a modified biomimetic deposition method to remineralize the surface of etched enamel to form mineral layers containing organized needle-like fluoridated hydroxyapatite crystals. The effect of a recombinant amelogenins (rP172) on the microstructure of the mineral in the coating was analyzed by SEM, XRD and FT-IR. At rP172 concentrations below 33 μg/mL, no significant effect was observed. In the presence of 1 mg/L F and at a concentration of 33 μg/mL rP172, formation of fused crystals growing from the enamel surface was initiated. Amelogenin promoted the oriented bundle formation of needle-like fluoridated hydroxyapatite in a dose dependent manner. Biomimetic synthesis of the amelogenin fluoridated hydroxyapatite nano-composite is one of the primary steps towards the development and design of novel biomaterial for future application in reparative and restorative dentistry.

Amelogenin (Amel) accelerates the nucleation of hydroxyapatite (HAP) in supersaturated solutions of calcium phosphate (Ca-P), shortening the induction time (delay period), under near-physiological conditions of pH, temperature, and ionic strength. Hierarchically organized Amel and amorphous calcium phosphate (ACP) nanorod microstructures are formed involving co-assembly of Amel-ACP particles at low supersaturations and low protein concentrations in a slow, well-controlled, constant composition (CC) crystallization system. At the earliest nucleation stages, the CC method allows the capture of prenucleation clusters and intermediate nanoclusers, spherical nanoparticles, and nanochains prior to enamel-like nanorod microstructure formations at later maturation stages. Amel-ACP nanoscaled building blocks are formed spontaneously by synergistic interactions between flexible Amel protein molecules and Ca-P prenucleation clusters, and these spherical nanoparticles evolve by orientated aggregation to form nanochains. Our results suggest that, in vivo, Amel may determine the structure of enamel by controlling prenucleation cluster aggregation at the earliest stages by forming stable Amel-ACP microstructures prior to subsequent crystal growth and mineral maturation.