Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health , Bethesda, Maryland 20892-0520, United States.
Department of Biochemistry, University of Zurich , Winterthurerstrasse 190, 8057 Zurich, Switzerland.
J Am Chem Soc. 2016 Sep 14;138(36):11702-13. doi: 10.1021/jacs.6b05443. Epub 2016 Sep 1.
Chemical denaturants are the most commonly used agents 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. Studies using Förster resonance energy transfer (FRET) and other methods found an increase in radius of gyration with denaturant concentration, but most small-angle X-ray scattering (SAXS) studies found 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 favorable 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 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.
化学变性剂是最常用的使蛋白质变性的试剂,被认为通过更好地溶解变性状态来发挥作用。随着变性剂浓度的增加,预计更好的溶剂化作用会导致变性链的扩展,从而提供对变性剂作用的敏感探针。然而,到目前为止,关于化学变性的影响,实验得到了定性上不同的结果。使用Förster 共振能量转移(FRET)和其他方法的研究发现,随着变性剂浓度的增加,回转半径增大,但大多数小角 X 射线散射(SAXS)研究发现没有变化。因此,这种差异挑战了我们对变性机制的理解,更普遍地挑战了这些实验应用于变性或无序蛋白质的准确性。在这里,我们使用全原子分子模拟来研究脲和盐酸胍对内在无序蛋白 ACTR 结构的影响,该蛋白可以通过实验在广泛的变性剂浓度范围内进行研究。使用带有精心校准的变性剂模型的无偏分子模拟,我们发现随着变性剂浓度的增加,蛋白质链确实会膨胀。这是由于脲或盐酸胍与所有残基的骨架以及几乎所有残基的侧链的有利缔合,从这种缔合推断出的变性剂-水转移自由能与实验估计值相当吻合。变性剂与骨架的相互作用主要由氢键主导,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 效率和 SAXS 强度,我们表明模拟轨迹与该蛋白质的实验一致,表明这两种类型的实验之间没有根本的不一致。与实验的一致性也支持了我们在模拟中描述的化学变性图景,由变性剂与蛋白质的弱缔合驱动。我们的模拟支持了每个实验所需的一些假设,以准确反映蛋白质大小的变化,即常用的 FRET 发色团不会从根本上改变结果,并且可能的影响,如优先溶剂分配到链的内部,不会干扰从 SAXS 实验确定回转半径。