Solid State and Structural Chemistry Unit, Indian Institute of Science , Bangalore, Karnataka 560012, India.
Department of Chemistry, University of Texas at Austin , Austin, Texas 78712, United States.
J Phys Chem B. 2017 Feb 9;121(5):995-1009. doi: 10.1021/acs.jpcb.6b13100. Epub 2017 Jan 25.
The folding of small protein ubiquitin (Ub), which plays an indispensable role in targeting proteins for degradation and DNA damage response, is complex. A number of experiments on Ub folding have reached differing conclusions regarding the relation between collapse and folding, and whether intermediates are populated. In order to resolve these vexing issues, we elucidate the denaturant-dependent thermodynamics and kinetics of Ub folding at low and neutral pH as a function of guanidinium chloride and urea using coarse-grained molecular simulations. The changes in the fraction of the folded Ub, and the radius of gyration (R) as a function of the denaturant concentration, [C], are in quantitative agreement with experiments. Under conditions used in experiments, R of the unfolded state at neutral pH changes only by ≈17% as the [GdmCl] decreases from 6 to 0 M. We predict that the extent of compaction of the unfolded state increases as temperature decreases. A two-dimensional folding landscape as a function of R and a measure of similarity to the folded state reveals unambiguously that the native state assembly is preceded by collapse, as discovered in fast mixing experiments on several proteins. Analyses of the folding trajectories, under mildly denaturing conditions ([GdmCl] = 1.0 M or [Urea] = 1.0 M), shows that Ub folds by collision between preformed secondary structural elements involving kinetic intermediates that are primarily stabilized by long-range contacts. Our work explains the results of small angle X-ray scattering (SAXS) experiments on Ub quantitatively, and establishes that evolved globular proteins in the unfolded ensemble are poised to collapse as the solvent conditions for the biopolymer changes from good solvent to Θ-solvent like conditions on denaturant dilution. In the process, we explain the discrepancy between SAXS and single molecule fluorescent resonant energy transfer (smFRET) experiments, which have arrived at a contradicting conclusion concerning the collapse of polypeptide chains.
小分子蛋白泛素（Ub）的折叠在蛋白质靶向降解和 DNA 损伤反应中起着不可或缺的作用，其过程非常复杂。许多关于 Ub 折叠的实验在折叠和崩溃之间的关系以及中间产物是否存在等问题上得出了不同的结论。为了解决这些棘手的问题，我们使用粗粒分子模拟阐明了在低 pH 和中性 pH 下，Ub 折叠的变性剂依赖性热力学和动力学特性，以及胍盐酸盐和尿素的影响。折叠 Ub 分数和回转半径（R）随变性剂浓度[C]的变化与实验结果定量一致。在实验中使用的条件下，中性 pH 下未折叠状态的 R 仅当[GdmCl]从 6 M 降低至 0 M 时才变化约 17%。我们预测，随着温度的降低，未折叠状态的紧凑程度会增加。作为 R 的函数的二维折叠景观以及与折叠状态的相似性度量明确表明，天然状态的组装是由崩溃引发的，这与几种蛋白质的快速混合实验结果一致。在轻度变性条件下（[GdmCl]=1.0 M 或[Urea]=1.0 M）分析折叠轨迹的结果表明，Ub 通过涉及涉及长程相互作用的预形成二级结构元件之间的碰撞折叠，这是动力学中间体的主要稳定因素。我们的工作定量解释了 Ub 的小角 X 射线散射（SAXS）实验结果，并确定了在从良溶剂到变性剂稀释时类似于θ溶剂的溶剂条件下，天然状态下的进化球状蛋白质处于崩溃的边缘。在这个过程中，我们解释了 SAXS 和单分子荧光共振能量转移（smFRET）实验之间的差异，后者在多肽链的崩溃问题上得出了相互矛盾的结论。
