Fluorescence indicates hierarchical amelogenin self-assembly

Amelogenin is the major protein component of developing enamel and guides enamel crystallization. Amelogenin is known to self-assemble into nanospheres, but hierarchical assembly into elongated structures is suggested due to the fibrillar nature of apatite crystals in enamel. Objective: To investigate self assembly of full-length recombinant human amelogenin (rH175) into supramolecular structures using fluorescence spectroscopy. Methods: The fluorescence intensity of solutions containing 0.5 mg/ml was studied using a fluorescence spectroscopy plate reader at 37ºC. An excitation wavelength of 280 nm was used and emission spectra between 300 and 500 nm collected. In a second set, an excitation wavelength at 450 nm was used and emission recorded at 482 nm. Various chemical conditions were investigated for a period of up to 7 days, including pH at 3.0, 5.5 6.5, 7.0 and 8.5; additions of 147 mM KCl, 1.6 mM calcium and 1.0 mM phosphate. Atomic force microscopy was used to study tertiary structure of protein self-assembly over time. Results: At 280 nm, tryptophan fluorescence maxima were observed at 348 nm at low pH, but shifted to ~340 nm when pH was raised to 6.0. In the presence of phosphate the shift occurred at pH 7. Solution at pH 7 and above showed a reversal of the fluorescence maxima to 348 nm after seven days. A progressive increase of fluorescence was observed in the first forty eight hours and a constant fluorescence was maintained subsequently. AFM showed the presence of short strings composed of amelogenin nanospheres after seven days at 37ºC. Conclusion: Change in fluorescence at 348 nm is indicative of changes in orientation of tryptophan from the surface to the core of the protein. This change is reversed over time indicating a reorganization of the protein which may be related to a hierarchical self-assembly process.

Support: by NIH/NIDCR R01-DE017529 and R01-DE015821