Dissecting Amelogenin Protein Nanospheres CHARACTERIZATION OF METASTABLE OLIGOMERS
Dissecting Amelogenin Protein Nanospheres CHARACTERIZATION OF METASTABLE OLIGOMERS
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DOI：
10.1074/jbc.m111.250928
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发表时间：
2011-10-07
影响因子：
4.8
通讯作者：
Moradian-Oldak, Janet
中科院分区：
文献类型：
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作者：
Bromley, Keith M.;Kiss, Andrew S.;Moradian-Oldak, Janet
釉原蛋白自组装形成细胞外蛋白质基质，该基质作为持续生长的釉质磷灰石晶体的模板。为了进一步深入了解釉原蛋白纳米球形成的分子机制，我们通过改变pH值、温度和蛋白质浓度来操纵釉原蛋白单体之间的相互作用，以产生分离的亚稳态釉原蛋白寡聚体。使用了重组猪釉原蛋白（rP172和rP148）以及三种仅含单个色氨酸（色氨酸161、色氨酸45和色氨酸25）的不同突变体。动态光散射和荧光研究表明，寡聚体是亚稳态的，并且与单体处于恒定平衡状态。在pH 5.5、4 - 10 mg/ml之间观察到平均流体动力学半径（R - H）为7.5 nm的稳定寡聚体。我们没有发现单体自组装成寡聚体时折叠显著增加的任何证据，这表明它们是无序的。对含单个色氨酸的釉原蛋白进行的荧光实验表明，在寡聚化时，釉原蛋白的C末端（大约在色氨酸161残基周围）暴露在寡聚体表面，而色氨酸25和色氨酸45周围的N末端区域参与蛋白质 - 蛋白质相互作用。截短的rP148形成了类似但更小的寡聚体，这表明C末端对于釉原蛋白寡聚化不是关键的。我们提出了一个通过寡聚体形成纳米球的模型，并且预测在弱酸性环境中，通过组氨酸质子化，纳米球将分解形成寡聚体。我们进一步提出，寡聚体结构可能是釉质磷灰石成熟过程中的功能组分。
Amelogenin self-assembles to form an extracellular protein matrix, which serves as a template for the continuously growing enamel apatite crystals. To gain further insight into the molecular mechanism of amelogenin nanosphere formation, we manipulated the interactions between amelogenin monomers by altering pH, temperature, and protein concentration to create isolated metastable amelogenin oligomers. Recombinant porcine amelogenins (rP172 and rP148) and three different mutants containing only a single tryptophan (Trp(161), Trp(45), and Trp(25)) were used. Dynamic light scattering and fluorescence studies demonstrated that oligomers were metastable and in constant equilibrium with monomers. Stable oligomers with an average hydrodynamic radius (R-H) of 7.5 nm were observed at pH 5.5 between 4 and 10 mg.ml(-1). We did not find any evidence of a significant increase in folding upon self-association of the monomers into oligomers, indicating that they are disordered. Fluorescence experiments with single tryptophan amelogenins revealed that upon oligomerization the C terminus of amelogenin (around residue Trp161) is exposed at the surface of the oligomers, whereas the N-terminal region around Trp25 and Trp45 is involved in protein-protein interaction. The truncated rP148 formed similar but smaller oligomers, suggesting that the C terminus is not critical for amelogenin oligomerization. We propose a model for nanosphere formation via oligomers, and we predict that nanospheres will break up to form oligomers in mildly acidic environments via histidine protonation. We further suggest that oligomeric structures might be functional components during maturation of enamel apatite.