Probing the Action of Chemical Denaturant on an Intrinsically Disordered Protein by Simulation and Experiment.
Probing the Action of Chemical Denaturant on an Intrinsically Disordered Protein by Simulation and Experiment.
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DOI：
10.1021/jacs.6b05443
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发表时间：
2016-09-14
影响因子：
15
通讯作者：
Best RB
中科院分区：
文献类型：
--
作者：
Zheng W;Borgia A;Buholzer K;Grishaev A;Schuler B;Best RB
化学变性剂是最常用的蛋白质去折叠试剂，被认为是通过更好地溶解未折叠状态而起作用的。随着变性剂浓度的增加，改进的溶剂化有望导致未折叠的链的扩展，从而提供变性剂作用的灵敏探针。然而，到目前为止，关于化学变性的影响，实验得到了定性不同的结果，使用Förster共振能量转移(FRET)和其他方法的研究发现，旋转半径随变性剂浓度的增加而增加，但小角X射线散射(SAXS)研究发现几乎没有变化。因此，这种差异挑战了我们对变性机制的理解，更广泛地说，挑战了这些应用于未折叠或无序蛋白质的实验的准确性。在这里，我们使用全原子分子模拟来研究尿素和氯化胍对固有无序蛋白质ACTR结构的影响，这可以通过实验在广泛的变性剂浓度范围内进行研究。使用无偏分子模拟和精心校准的变性剂模型，我们发现蛋白质链确实随着变性剂浓度的增加而膨胀。这是由于尿素或氯化胍与所有残基的骨架和几乎所有残基的侧链发生了有利的缔合，由此推导出的变性水转移自由能与实验估计合理地一致。变性剂与主链的相互作用主要以氢键为主，而与侧链的相互作用包括其他贡献。通过计算每个变性剂浓度下的FRET转移效率和SAXS强度，我们发现模拟轨迹与该蛋白质的两个实验都是一致的，说明两种实验之间没有根本的不一致。与实验的一致也支持我们模拟中描述的化学变性的画面，这是由变性剂与蛋白质的弱结合所驱动的。我们的模拟支持每个实验准确反映蛋白质大小变化所需的一些假设，即常用的FRET生色团不会定性地改变结果，并且可能的影响，如优先分配到链的内部，不会干扰SAXS实验中旋转半径的确定。
Chemical denaturants are the most commonly used agent for unfolding proteins, and are thought to act by better solvating the unfolded state. Improved solvation is expected to lead to an expansion of unfolded chains with increasing denaturant concentration, providing a sensitive probe of the denaturant action. However, experiments have so far yielded qualitatively different results concerning the effects of chemical denaturation, with studies using Förster resonance energy transfer (FRET) and other methods finding an increase in radius of gyration with denaturant concentration, but with small-angle X-ray scattering (SAXS) studies finding mostly no change. This discrepancy therefore challenges our understanding of denaturation mechanism, and more generally the accuracy of these experiments as applied to unfolded or disordered proteins. Here, we use all-atom molecular simulations to investigate the effect of urea and guanidinium chloride on the structure of the intrinsically disordered protein ACTR, which can be studied by experiment over a wide range of denaturant concentration. Using unbiased molecular simulations with a carefully calibrated denaturant model, we find that the protein chain indeed swells with increasing denaturant concentration. This is due to the favourable association of urea or guanidinium chloride with the backbone of all residues and with the side-chains of almost all residues, with denaturant-water transfer free energies inferred from this association in reasonable accord with experimental estimates. Interactions of the denaturants with the backbone are dominated by hydrogen bonding, while interactions with side-chains include other contributions. By computing FRET transfer efficiencies and SAXS intensities at each denaturant concentration, we show that the simulation trajectories are in accord with both experiments on this protein, demonstrating that there is no fundamental inconsistency between the two types of experiment. Agreement with experiment also supports the picture of chemical denaturation described in our simulations, driven by weak association of denaturant with the protein. Our simulations support some assumptions needed for each experiment to accurately reflect changes in protein size, namely that the commonly used FRET chromophores do not qualitatively alter the results, and that possible effects such as preferential solvent partitioning into the interior of the chain do not interfere with the determination of radius of gyration from the SAXS experiments.
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影响因子：
16.6
作者：
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影响因子：
64.8
作者：
Demarest, SJ;Martinez-Yamout, M;Wright, PE
通讯作者：
Wright, PE
DOI：
10.1073/pnas.91.5.1746
发表时间：
1994-03-01
影响因子：
11.1
作者：
CAFLISCH, A;KARPLUS, M
通讯作者：
KARPLUS, M
影响因子：
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作者：
Graen, Timo;Hoefling, Martin;Grubmueller, Helmut
通讯作者：
Grubmueller, Helmut
DOI：
10.1073/pnas.090104997
发表时间：
2000-05-09
影响因子：
11.1
作者：
Deniz, AA;Laurence, TA;Weiss, S
通讯作者：
Weiss, S