Characterization of Amelogenin Nanospheres With Analytical Ultracentrifugation

Objectives: Amelogenin is a major enamel protein and is essential in regulating mineral formation. Amelogenin is known to self-assemble into supramolecular complexes termed nanospheres. These structures further assemble into a matrix which serves as a scaffold for developing enamel ribbons. However, information about the hydrodynamic properties and composition of these nanospheres prompts further structural characterization. The goal was to study the effects of pH, protein concentration and incubation time on amelogenin nanosphere formation using analytical ultracentrifugation.
Methods: Recombinant murine amelogenin (rM179) was expressed from pET179 in E. coli and purified by HPLC as described earlier (Simmer, 1994). rM179 was dissolved in a buffer containing 10 mM sodium phosphate, 5 mM sodium chloride and 2 mM octylglucoside. rM179 samples were centrifuged individually at 25,000-50,000 rpm using a Beckman XL-A with absorbance optics at 230 nm at OD 0.3 and 0.9. The sedimentation velocity and hydrodynamic data were analyzed with the UltraScan3 ver. 3.4 software at high resolution using high-performance computing modules for 2-dimensional spectrum analysis and Monte Carlo analysis.
Results: rM179 remained monomeric with sedimentation coefficients of 1.3-1.8 S at pH 4.4-6.4, aggregated at pH 6.6-7.8, and formed heterogeneous complexes of 30-300 S at pH 8.0-8.2. At pH 8.6-9.0 homogeneous 20-37 S species were observed (20 nm in diameter). Frictional ratios of f/f0 = 1.0-1.45 indicated the existence of spherical and elongated species respectively. While high rM179 concentrations promoted the formation of smaller species (22-26 S), low concentrations favor larger species (19-32 S). rM179 assemblies “ripened” into their final form over time. Observations under transmission electron microscopy validated the formation of assemblies.
Conclusions: For the first time, analytical ultracentrifugation was used to determine the hydrodynamic properties of rM179 assemblies. We also conclude there is coexistence of nanospheres and elongated assemblies, and time and mass-action dependency of rM179 assembly formation.