The Tooth Enamel Protein, Porcine Amelogenin, Is an Intrinsically Disordered Protein with an Extended Molecular Configuration in the Monomeric Form

Abstract

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...

Probing the self‐association, intermolecular contacts, and folding propensity of amelogenin

“…The aqueous rP172 sample features only a single (-) pi-pi* transition band centered at 203 nm that is representative of a predominantly disordered protein that contains random coil (RC) and other secondary structure elements. 9 However, as TFE content increases, we note a corresponding increase in alpha-helical content as evidenced by the emergence of pi-pi* and n-pi* transition bands at 208 and 222 nm, respectively, 9 with a maximum effect achieved at 30% TFE. This CD dataset demonstrates that in the presence of TFE, certain regions within the rP172 protein fold into helical structures.…”

“…[1][2][3] The major protein component, amelogenin, is essential for normal enamel development and forms supramolecular assemblies (nanospheres) 4,5 that are believed to exert control over the mineral phase, morphology, organization, and directionality of hydroxyapatite crystal growth. 6,7 Recent bioinformatics and biophysical studies 8,9 have demonstrated that several amelogenins have sequence characteristics that fit the profile of intrinsically disordered proteins or IDPs. [9][10][11][12][13][14] The existence of unstructured regions within monomeric amelogenin have been confirmed by solution NMR studies of two recombinant amelogenins, murine 15 and porcine, 9 as well as model peptides representing amelogenin polyproline Type II (PPII) repeat regions.…”

“…6,7 Recent bioinformatics and biophysical studies 8,9 have demonstrated that several amelogenins have sequence characteristics that fit the profile of intrinsically disordered proteins or IDPs. [9][10][11][12][13][14] The existence of unstructured regions within monomeric amelogenin have been confirmed by solution NMR studies of two recombinant amelogenins, murine 15 and porcine, 9 as well as model peptides representing amelogenin polyproline Type II (PPII) repeat regions. 16 In the case of the porcine species, under low pH aqueous conditions the monomeric form of amelogenin exists in an extended, unfolded state.…”

“…16 In the case of the porcine species, under low pH aqueous conditions the monomeric form of amelogenin exists in an extended, unfolded state. 9 It is suspected that intrinsic disorder contributes to the structure and function of these proteins in self-assembly 5,9,15,[17][18][19][20][21][22] and in cellmatrix, protein-matrix, and protein-mineral interactions. [5][6][7][8][9][23][24][25] The main challenge is to elucidate how intrinsic disorder affects the molecular behavior and configuration of this unusual series of proteins, and how amelogenin molecular behavior, in turn, affects biomineralization within the enamel matrix.…”

“…9 These are the highly conserved N-terminus (P2-W45) 26 or the proposed selfassembly ''A domain'' (P2-M42), [20][21][22] the partially condensed, charged C-terminal domain (D155-D173), 9,[20][21][22] and the extended Pro, Met, Gln-rich central domain (T58-P154) 9,16 that contains the polyproline Type II I70-P89 and P102-P145 sequence regions. 9 Both terminal domains exhibited evidence of residual secondary structure, 9 and these terminal regions may represent putative regions for folding during amelogenin-target interactions or self-assembly. 27 It is experimentally challenging to assess the folding propensities of the three major regions of porcine amelogenin in the presence of matrix targets or during the self-assembly process.…”