The cooperative self-assembly of 25 and 23kDa amelogenins.

Self-assembly of the extracellular matrix protein amelogenin is believed to play an essential role in regulating the growth and organization of enamel crystals during enamel formation. The full-length amelogenin uniquely regulates the growth, shape, and arrangement of enamel crystals. Protein hydrolysis will ultimately facilitate a tissue with high mineral content. Protein processing is however highly specific suggesting a functional role of the cleaved amelogenins in enamel maturation. Here we hypothesize that the cooperative self-assembly of the recombinant full-length amelogenin 25kDa and the 23kDa proteolytic cleavage product is a function of pH, mixing ratio and incubation time and is associated with the isoelectric point of the protein. Self-assembly of amelogenin into nanospheres which increased in size with increasing pH was observed by atomic force microscopy. Elongated structures of about 100nm length and 25nm width formed over several days for amelogenin 25 and 23kDa predominantly at pH-values of 6.5 and 7.5, respectively. When both proteins 25 and 23kDa were mixed, self-assembled nanostrings of 200-300nm length consisting of fused nanospheres were obtained at pH around 7.0 within 24h. The protein nanostrings formed links over time and a continuous mesh was obtained after 7 days. Electrical conductivity data also showed gradual changes when both amelogenins were mixed in solutions supporting the idea that elongated structures form over extended periods of time. We propose that due to the difference in the isoelectric point, self-assembled nanospheres composed of 23 or 25kDa amelogenin have opposite ionic charges at pH-values around 7.0 and thus experience ionic attraction that enables cooperative self-assembly.