The leucine-rich amelogenin protein (LRAP) is primarily monomeric and unstructured in physiological solution

Abstract

Amelogenin proteins are critical to the formation of enamel in teeth and may have roles in controlling growth and regulating microstructures of the intricately woven hydroxyapatite (HAP). Leucine-rich amelogenin protein (LRAP) is a 59-residue splice variant of amelogenin and contains the N- and C-terminal charged regions of the full-length protein thought to control crystal growth. Although the quaternary structure of full-length amelogenin in solution has been well studied and can consist of self-assemblies of monomers called nanospheres, there is limited information on the quaternary structure of LRAP. Here, sedimentation velocity analytical ultracentrifugation (SV) and small angle neutron scattering (SANS) were used to study the tertiary and quaternary structure of LRAP at various pH values, ionic strengths, and concentrations. We found that the monomer is the dominant species of phosphorylated LRAP (LRAP(+P)) over a range of solution conditions (pH 2.7-4.1, pH 4.5-8, 50 mmol/L(mM) to 200 mM NaCl, 0.065-2 mg/mL). The monomer is also the dominant species for unphosphorylated LRAP (LRAP(-P)) at pH 7.4 and for LRAP(+P) in the presence of 2.5 mM calcium at pH 7.4. LRAP aggregates in a narrow pH range near the isoelectric point of pH 4.1. SV and SANS show that the LRAP monomer has a radius of ∼2.0 nm and an asymmetric structure, and solution NMR studies indicate that the monomer is largely unstructured. This work provides new insights into the secondary, tertiary, and quaternary structure of LRAP in solution and provides evidence that the monomeric species may be an important functional form of some amelogenins.

Keywords: Amelogenin; Biomineralization; Enamel; LRAP; Nanosphere.

Figure 1

Primary amino acid sequence of murine amelogenin with the basic and acidic residues colored blue and red, respectively. The splice variant LRAP is composed of the N-terminal 33 and C-terminal 26 residues, shaded yellow. Both full-length amelogenin and LRAP are post-translationally modified by side chain phosphorylation of S16, colored green. The serine at position 16 was either phosphorylated (LRAP(+P)) or non-phosphorylated (LRAP(−P)).

Figure 2

Zeta potential of LRAP (+P) in SCP solution at 50 and 150 mM NaCl as a function of pH. Data points are connected by smoothed lines.

Figure 3

Normalized sedimentation coefficient distribution for LRAP(+P) in 150 mM NaCl, pH 7.4 SCP solution at 2 mg/mL with a 400x expansion in the inset. Peaks are labeled with % amount and sedimentation coefficient, s_20,w, value in svedbergs (S). The main peak was determined to represent a monomer and the s_20,w value of the monomer peak is the average over the peak.

Figure 4

a) R_h for LRAP(+P) species in 0.065 mg/ml 50 mM NaCl SCP solutions including R_h of monomers determined by SV (open blue circles) and R_h of aggregates at pH 4.2 determined by DLS (filled blue circle), b) SV-determined f/f₀ ratios for monomers in 0.065 mg/mL LRAP solutions including LRAP(+P) in 50 mM NaCl SCP (blue circles), LRAP(+P) in 150 mM NaCl SCP at pH 7.4 (green square), LRAP(−P) in 150 mM NaCl SCP at pH 7.4 (red triangle), and LRAP(+P) in 2.5 mM CaCl₂ at pH 7.4 (orange diamond).

Figure 5

SANS I(q) vs. q data for a) 1 mg/mL LRAP(+P) in 2% acetic acid (pH 2.7) (black) and 150 mM NaCl, SCP (pH 7.4) (red) and b) 2 mg/mL (blue) and 1 mg/mL (black) LRAP(+P) in 2% acetic acid (pH 2.7) with the 2 mg/mL data normalized to the 1 mg/mL data to allow better comparison of the curves.

Figure 6

a) The SANS data from 2 mg/mL LRAP(+P) in 2% acetic acid (pH 2.7) along with the corresponding average (red) and best-fit (blue) model trace from the SASSIE calculations. The best-fit (blue) LRAP(+P) structure is shown under the traces. b) A χ² versus R_g plot for the ensemble of 9427 SASSIE structures derived from the SANS data for 2 mg/mL LRAP(+P) in 2% acetic acid (pH 2.7). The corresponding points for the 10 lowest energy Rosetta model structures are shown in red. The best-fit (black) SASSIE structure and best-fit (red) lowest energy Rosetta structure are superimposed above the traces.

Figure 7

Analysis of the observed ¹³C^α and ¹³C^β chemical shift deviations from random coil values for LRAP(−P) in 150 mM NaCl, SCP solution, pH 7.4 where Δδ¹³C = δ_{Random coil}-δ_Observed. Red = ¹³C^α, cyan = ¹³C^β. Asterisks indicate residues where it was not possible to obtain unambiguous ¹³C^β assignments. The dashed purple line separated the residues present in the N- and C-terminal ends of full-length murine amelogenin. Random coil values obtained from cns_solve_1.1.