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...