Osmolyte-Induced Folding of an Intrinsically Disordered Protein: Folding Mechanism in the Absence of Ligand
Osmolyte-Induced Folding of an Intrinsically Disordered Protein: Folding Mechanism in the Absence of Ligand
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DOI：
10.1021/bi100222h
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发表时间：
2010-06-29
期刊：
影响因子：
2.9
通讯作者：
Oas, Terrence G.
中科院分区：
文献类型：
--
作者：
Chang, Yu-Chu;Oas, Terrence G.
理解蛋白质折叠过程中存在的热力学可分辨状态之间的相互转换不仅可以提供蛋白质折叠的动力学和能量学，而且还可以深入了解这些状态在生物系统中的功能作用。枯草芽孢杆菌的细菌核糖核酸酶P全酶的蛋白质组分(P蛋白)在缺乏同源RNA或其他阴离子配体的情况下被展示出来。本研究以P蛋白为模型系统，探讨固有无序蛋白(IDP)折叠机制的一般特征。三甲胺N-氧化物(TMAO)是一种稳定蛋白质未连接折叠形式的渗透分子，它的使用使我们能够在没有配体的情况下研究P蛋白的折叠过程。不同最终TMAO浓度下的瞬时停流动力学曲线表现为多相动力学。在滴定过程中，分别用TMAO和尿素进行平衡“共滴定”实验，得到尿素-TMAO滴定的P蛋白表面。动力学和平衡研究都表明，在P蛋白折叠过程中存在一种以前未被检测到的中间状态。中间状态显著填充，与大小和拓扑相似的固有折叠蛋白质相比，折叠速度常数相对较慢。所述的实验和分析为其他国内流离失所者的机械折叠研究提供了有益的范例。
Understanding the interconversion between thermodynamically distinguishable states present in a protein folding pathway provides not only the kinetics and energetics of protein folding but also insights into the functional roles of these states in biological systems. The protein component of the bacterial RNase P holoenzyme from Bacillus subtilis (P protein) was previously shown to be unfolded in the absence of its cognate RNA or other anionic ligands. P protein was used in this study as a model system to explore general features of intrinsically disordered protein (IDP) folding mechanisms. The use of trimethylamine N-oxide (TMAO), an osmolyte that stabilizes the unliganded folded form of the protein, enabled us to study the folding process of P protein in the absence of ligand. Transient stopped-flow kinetic traces at various final TMAO concentrations exhibited multiphasic kinetics. Equilibrium "cotitration" experiments were performed using both TMAO and urea during the titration to produce a urea-TMAO titration surface of P protein. Both kinetic and equilibrium studies show evidence of a previously undetected intermediate state in the P protein folding process. The intermediate state is significantly populated, and the folding rate constants are relatively slow compared to those of intrinsically folded proteins similar in size and topology. The experiments and analysis described serve as a useful example for mechanistic folding studies of other IDPs.