Differences in Self-Assembly Between Native and Recombinant Full-Length Porcine Amelogenin

Recent in vitro findings have suggested that full-length amelogenins self-assemble to form higher order structures that potentially guide the formation and organization of developing enamel crystals. Such studies have been predominantly carried out using recombinant proteins lacking one N-terminal methionine and the single phosphate group (S-16). Objective: This study was carried out to determine if such differences in composition affect protein self-assembly under conditions of physiological pH, temperature, and ionic strength, using full-length recombinant (rP172) and native (P173) porcine amelogenins. Methods: Protein samples (2mg/mL) prepared in 200mM TRIS/HCl buffer (adjusted to pH 7.2 at 37 șC; IS = 163mM), were studied using dynamic light scattering (DLS) and TEM. DLS data were collected between pH 8 and pH 7.1, by slowly changing buffer pH by increasing temperature from 7 șC to 43 șC. For TEM analyses, 5 µL samples (pH 7.2 at 37 șC) were incubated on TEM grids placed in a humidity chamber at 37 șC. Incubation was stopped after 40 minutes and samples were negatively stained with 1% PTA, pH 7.2. Results: Changes in self-assembly (indicated by marked increases in DLS scattering) of P173 occurred at significantly higher pH (pH 7.8) than for rP172 (pH 7.2). Although both proteins exhibited spherical nanoparticles that aligned to form elongated structures, as viewed by TEM, there were notable differences between these assemblies and in the size of the subunits. The width of the elongated structures of P173 (7.8 ± 1.7nm, N=180) were significantly (p= 0.01) narrower than those in similar structures in rP172 (10.1 ± 2.2 nm, N=180). The P173 assemblies also appeared to form a more branched and denser network. Conclusion: Although both recombinant and native porcine amelogenins assemble into chain-like branched and interconnected structures, noted differences in composition significantly affect self-assembly behavior. Supported by NIDCR grants DE016376 and DE007327.