Towards a complete description of the structural and dynamic properties of the denatured state of barnase and the role of residual structure in folding.
Towards a complete description of the structural and dynamic properties of the denatured state of barnase and the role of residual structure in folding.
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DOI：
10.1006/jmbi.2000.3523
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发表时间：
2000-03
影响因子：
5.6
通讯作者：
Kam-Bo Wong;J. Clarke;C. J. Bond;J. Neira;S. Freund;A. Fersht;V. Daggett
中科院分区：
文献类型：
--
作者：
Kam-Bo Wong;J. Clarke;C. J. Bond;J. Neira;S. Freund;A. Fersht;V. Daggett
由于变性蛋白质的流动性和构象异质性，其详细表征仍然是难以捉摸的。核磁共振研究开始提供有关变性状态下残余结构的线索，但所得数据过于稀疏，无法使用常规技术转化为分子模型。分子动力学模拟可以补充核磁共振提供详细的结构信息的组件的变性合奏。在这里，我们描述了三个独立的4纳秒高温分子动力学模拟barnase在水中。模拟的变性状态是构象异质性相对于一个单一的模拟和模拟之间填充的构象。尽管如此，有一些持续的相互作用，发生在不同程度上在所有的模拟，主要涉及流体疏水簇的形成与参与残基随时间的推移而变化。β（3-4）发夹的区域含有特别高程度的这种侧链相互作用，但在三种变性集合中的两种中缺乏β结构：β（3-4）是β结构中唯一在变性状态下含有显著残留结构的部分。两个主要的α-螺旋（α 1和α 2）采用动态螺旋结构。此外，还有持久的接触，从蛋白质的主体中夹断核心2。蛋白质的其余部分是非结构化的，除了短暂的和主要是局部的侧链相互作用。总的来说，模拟变性状态包含残留结构的形式，动态，波动的二级结构在α 1和α 2，以及波动的三级接触在β（3-4）区域，和α 1和β（3-4）之间，与以前的NMR研究。在这里，我们还表明，这些地区含有残余结构显示受损的流动性的分子动力学和NMR弛豫实验。残余结构在减少链的构象状态和修复断裂区域方面很重要。例如，β（3-4）和α 1之间的三级接触有助于α 1的重折叠。这种接触辅助的螺旋形成在单独和复合的β（3-4）和α 1的片段模拟中得到证实，因此，α 1和β（3-4）似乎是折叠起始位点。这些网站在折叠中的作用进行了研究，向后工作，并考虑在反向模拟，注意到较早的时间点从模拟提供的主要中间和过渡态的模型在定量协议的数据从展开和重折叠实验。β（3-4）和α 1在变性状态下都是动态的，但当它们碰撞并产生足够的接触时，它们提供了一个松散的结构支架，进一步的β链堆积在上面。β-结构在β（3-4）附近凝聚，而α 1有助于稳定β（3-4）并保持其取向。所得的β-结构是相对平坦的，并且在主要中间体中是松散的。进一步的包装包封，结果β-折叠扭曲，导致主要的过渡态。该结构仍处于扩张状态，此时环未良好形成。然后发生填充相互作用的微调和结构的最终缩合以产生天然状态。
The detailed characterization of denatured proteins remains elusive due to their mobility and conformational heterogeneity. NMR studies are beginning to provide clues regarding residual structure in the denatured state but the resulting data are too sparse to be transformed into molecular models using conventional techniques. Molecular dynamics simulations can complement NMR by providing detailed structural information for components of the denatured ensemble. Here, we describe three independent 4 ns high-temperature molecular dynamics simulations of barnase in water. The simulated denatured state was conformationally heterogeneous with respect to the conformations populated both within a single simulation and between simulations. Nonetheless, there were some persistent interactions that occurred to varying degrees in all simulations and primarily involved the formation of fluid hydrophobic clusters with participating residues changing over time. The region of the beta(3-4) hairpin contained a particularly high degree of such side-chain interactions but it lacked beta-structure in two of the three denatured ensembles: beta(3-4) was the only portion of the beta-structure to contain significant residual structure in the denatured state. The two principal alpha-helices (alpha1 and alpha2) adopted dynamic helical structure. In addition, there were persistent contacts that pinched off core 2 from the body of the protein. The rest of the protein was unstructured, aside from transient and mostly local side-chain interactions. Overall, the simulated denatured state contains residual structure in the form of dynamic, fluctuating secondary structure in alpha1 and alpha2, as well as fluctuating tertiary contacts in the beta(3-4) region, and between alpha1 and beta(3-4), in agreement with previous NMR studies. Here, we also show that these regions containing residual structure display impaired mobility by both molecular dynamics and NMR relaxation experiments. The residual structure was important in decreasing the conformational states available to the chain and in repairing disrupted regions. For example, tertiary contacts between beta(3-4) and alpha1 assisted in the refolding of alpha1. This contact-assisted helix formation was confirmed in fragment simulations of beta(3-4) and alpha1 alone and complexed, and, as such, alpha1 and beta(3-4) appear to be folding initiation sites. The role of these sites in folding was investigated by working backwards and considering the simulation in reverse, noting that earlier time-points from the simulations provide models of the major intermediate and transition states in quantitative agreement with data from both unfolding and refolding experiments. Both beta(3-4) and alpha1 are dynamic in the denatured state but when they collide and make enough contacts, they provide a loose structural scaffold onto which further beta-strands pack. The beta-structure condenses about beta(3-4), while alpha1 aids in stabilizing beta(3-4) and maintaining its orientation. The resulting beta-structure is relatively planar and loose in the major intermediate. Further packing ensues, and as a result the beta-sheet twists, leading to the major transition state. The structure is still expanded and loops are not well formed at this point. Fine-tuning of the packing interactions and the final condensation of the structure then occurs to yield the native state.