A Solution NMR Investigation into the Early Events of Amelogenin Nanosphere Self-Assembly Initiated with Sodium Chloride or Calcium Chloride

Abstract

Using solution-state NMR spectroscopy, new insights into the early events governing amelogenin supramolecular self-assembly have been identified using sodium chloride and calcium chloride to trigger the association. Two-dimensional 1 H-15 N HSQC spectra were recorded for 15 N-and 13 Clabeled murine amelogenin as a function of increasing NaCl and CaCl 2 concentration beginning with solution conditions of 2% acetic acid at pH 3.0, where amelogenin was monomeric. Residue specific changes in molecular dynamics, manifested by the reduction in intensity and disappearance of 1 H-15 N HSQC cross-peaks, were observed with the addition of either salt to the protein. With increasing NaCl concentrations, residues between T21 and R31 near the N-terminus were affected first, suggesting that these residues may initiate amelogenin dimerization, the first step in nanosphere assembly. At higher NaCl concentrations, more residues near the N-terminus (Y12-I51) were affected, and with further additions of NaCl, residues near the C-terminus (L141-T171) began to show a similar change in molecular dynamics. With increasing CaCl 2 concentrations, a similar stepwise change in molecular dynamics involving essentially the same set of amelogenin residues was observed. As the concentration of either salt was increased, a concomitant increase in the estimated overall rotational correlation time (τ c ) was observed, consistent with assembly. Selfassembly into a dimer or trimer was established with dynamic light scattering studies under similar conditions that showed an increase in diameter of the smallest species from 4.1 nm in the absence of salt to ~10 nm in the presence of salt. These results suggest a possible stepwise interaction mechanism, starting with the N-terminus and followed by the C-terminus, leading to amelogenin nanosphere assembly.Dental enamel, the layer of elongated carbonated hydroxayapatite on the outer layer of the tooth, is the hardest tissue in the human body (1). It needs to be strong because it is exposed to repeated masticatory, parafunctional, and occasional impact loading, but unlike the dominant biomineral in the human body (2), mesenchyme-derived bone, enamel cannot self-repair nor can it undergo remodeling, and therefore, it must last a lifetime. Enamel's resistance to wear and deformation are due to a combination of high mineral content and unique three-dimensional structural organization (3). Ninety-five percent of mature enamel consists of long and narrow crystals of carbonated hydroxyapatite packed into parallel arrays, called enamel rods, that are intricately interwoven into an unique lattice architecture (4,5). There is little, if any, matrix

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

“…1(B)]. On the other hand, the 15 Na spins, being directly bonded to both the carbonyl and alpha-carbon atoms of the peptide backbone, are sensitive not only to solvent-induced chemical exchange at NH sites but also conformational events brought about by the solvent-stabilization effects of TFE alcohol binding along the polypeptide backbone [ Fig. 1(B)].…”

“…From previous NMR studies of salt, Ca (II)-induced amelogenin dimerization, it is known that the reduction in intensity and attenuation of 15 N-1 H HSQC crosspeaks occur due to conformational exchange. 15 Selective loss or broadening of NMR signals was also observed in aggregation studies involving various proteins in buffer salt environments 34 or TFE. 28 Thus, the attenuation of HSQC crosspeaks reflects TFEinduced protein-protein interactions involving intermolecular contacts with residues in the P4-H6, V19-P33, and E40-T58 regions of the N-terminus, and to a minor extent, short sequence regions (P84-M86, P141, P144-P145) within the central domain ( Fig.…”

“…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.…”

“…One of the major reasons for this is that the enamel matrix is a complex environment [2][3][4][5][6][20][21][22][23][24][25] and the true identity of amelogenin-specific targets, solution conditions, and other important features are not known at present. The other reason is that the amelogenin oligomerization process itself is quite complex 5,15,18,[20][21][22][23]27 and results in the formation of high-order high-molecular weight complexes that are difficult to study with conventional structural techniques such as NMR. To circumvent this problem, we turn to techniques and approaches that can offer insights into the native folding of IDPs and create conditions that mimic the self-assembly process in a more tractable fashion.…”