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 1H-15N HSQC spectra were recorded for 15N- and 13C-labeled murine amelogenin as a function of increasing NaCl and CaCl2 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 1H-15N 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 CaCl2 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 (tau(c)) was observed, consistent with assembly. Self-assembly 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.

FIGURE 1

(A) Overlay of the ¹H–¹⁵N HSQC spectra of rp(H)M180 in 2% acetic acid, pH 3.0, in the absence of salt (red) and in the presence of 1500:1 molar excess CaCl₂ (blue). (B) Overlay of the ¹H–¹⁵N HSQC spectra of rp(H)M180 in the presence of 1500:1 (blue) and 3000:1 (magneta) molar excess CaCl₂ and NaCl, respectively. Spectra recorded at 20 °C at a ₁H resonance frequency of 750 (CaCl₂) and 800 (NaCl) MHz.

FIGURE 2

Plot of the amide resonances that start to disappear (open circles) or completely disappear (red circles) in the ¹H–¹⁵N HSQC spectrum of ¹⁵N-labeled rp(H)M180 at different salt/rp(H)M180 molar ratios (indicated on the left). Underneath is a schematic illustration of murine amelogenin with various regions of the protein highlighted (26). The first three N-terminal regions comprise the N-terminal TRAP (tyrosine-rich amelogenin peptide) region (magenta = protein-protein interaction region, green = lectin-like binding tri-tyrosine domain). The cyan colored region is a hydrophobic segment cleaved by enamelysin, and the blue colored region is the hydrophilic C-terminal mineral-binding domain. The dashed blue lines point to the region first affected by the addition of Ca²⁺, and the dashed red lines are the regions affected at higher salt concentrations. Not shown on the plot are six residues that start to disappear (T63, F116, Q117, and H136) or have disappeared (A75 and Q83) at the highest sodium ion concentrations. Also not shown are two residues that disappear (T63 and M129) at the highest CaCl₂ concentration.

FIGURE 3

Dynamic light scattering data for rp(H)M180 (0.14 mM) prepared in 2% acetic acid (pH 3.0) with no salt (black solid line), 100 mM NaCl (red dashed line), and 50 mM CaCl₂ (blue dotted line). The increase in diameter with either salt is consistent with the self-association of amelogenin into larger complexes.

FIGURE 4

Plot of the hydrophilicity score for rp(H)M180 determined by the method of Hopp and Woods (52) using ProtScale software of ExPASy (Expert Protein Analysis System). Positive values are hydrophilic and negative values hydrophobic. On top is a schematic illustration of murine amelogenin with various regions highlighted according to the description in Figure 2.

FIGURE 5

Schematic showing the proposed stepwise interaction mechanism of amelogenin self-assembly into dimers. The monomers (left) first interact with each other near the N-terminus at intermediate salt concentrations (middle). At high salt concentrations (right), a larger region near the N-terminus and a region near the C-terminus interact. The monomers are drawn as elongated rods because the general consensus is that in the monomeric form much of the protein has the predisposition to form an extended β-sheet/β-strand and β-spiral/polyproline II (PPII) structure (5, 14, 15).