Department of Preventive and Restorative Dental Sciences, 707 Parnassus Ave., University of California, San Francisco, CA 94143-0758, USA.
J Struct Biol. 2008 Dec;164(3):314-21. doi: 10.1016/j.jsb.2008.09.005. Epub 2008 Sep 21.
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.
我们推测，重组全长釉原蛋白 25kDa 和 23kDa 蛋白水解产物的协同自组装是 pH、混合比和孵育时间的函数，并与蛋白质的等电点有关。原子力显微镜观察到釉原蛋白在 pH 值增加时自组装成纳米球，纳米球的直径随着 pH 值的增加而增大。对于 25kDa 和 23kDa 釉原蛋白，在 pH 值分别为 6.5 和 7.5 时，主要形成长达 100nm、宽 25nm 的伸长结构，需要数天时间。当两种蛋白 25 和 23kDa 混合时，在 pH 值约为 7.0 时，在 24 小时内得到由融合纳米球组成的 200-300nm 长的自组装纳米串。随着时间的推移，纳米串形成连接，7 天后获得连续的网格。当两种釉原蛋白混合在支持形成伸长结构的溶液中时，电导数据也显示出逐渐变化，这进一步支持了在较长时间内形成伸长结构的观点。我们提出，由于等电点的差异，由 23kDa 或 25kDa 釉原蛋白组成的自组装纳米球在 pH 值约为 7.0 时具有相反的离子电荷，因此会发生离子吸引，从而实现协同自组装。
