Gauthier Martin, Leclerc Jérémie, Lefèvre Thierry, Gagné Stéphane M, Auger Michèle
Department of Chemistry, Regroupement Québécois de Recherche sur la Fonction, la Structure et l'Ingénierie des Protéines (PROTEO), Centre de Recherche sur les Matériaux Avancés (CERMA), Centre Québécois sur les Matériaux Fonctionnels (CQMF), and ‡Department of Biochemistry, Microbiology and Bioinformatics, Institut de Biologie Intégrative et des Systèmes (IBIS), PROTEO, Université Laval , Québec, QC Canada , G1V 0A6.
Biomacromolecules. 2014 Dec 8;15(12):4447-54. doi: 10.1021/bm501241n. Epub 2014 Nov 4.
Spider silk proteins undergo a complex series of molecular events before being converted into an outstanding hierarchically organized fiber. Recent literature has underlined the crucial role of the C-terminal domain in silk protein stability and fiber formation. However, the effect of pH remains to be clarified. We have thus developed an efficient purification protocol to obtain stable native-like recombinant MaSp1 C-terminal domain of Nephila clavipes (NCCTD). Its structure was investigated as a function of pH using circular dichroism, fluorescence and solution NMR spectroscopy. The results show that the NCCTD structure is very sensitive to pH and suggest that a molten globule state occurs at pH 5.0 and below. Electronic microscopy images also indicate fiber formation at low pH and coarser globular particles at more basic pH. The results are consistent with a spinning process model where the NCCTD acts as an aggregation nucleus favoring the β-aggregation of the hydrophobic polyalanine repeats upon spinning.
蜘蛛丝蛋白在转化为出色的层次结构纤维之前会经历一系列复杂的分子事件。最近的文献强调了C末端结构域在丝蛋白稳定性和纤维形成中的关键作用。然而，pH值的影响仍有待阐明。因此，我们开发了一种高效的纯化方案，以获得稳定的、类似天然的大腹园蛛重组MaSp1 C末端结构域（NCCTD）。使用圆二色性、荧光和溶液核磁共振光谱研究了其结构随pH值的变化。结果表明，NCCTD结构对pH值非常敏感，表明在pH 5.0及以下会出现熔球状态。电子显微镜图像还显示在低pH值下会形成纤维，在碱性更强的pH值下会形成更粗大的球状颗粒。这些结果与一个纺丝过程模型一致，在该模型中，NCCTD作为聚集核，有利于纺丝时疏水聚丙氨酸重复序列的β-聚集。
