A half century of studying protein folding in vitro and modeling it in silico has not provided us with a reliable computational method to predict the native conformations of proteins de novo, let alone identify the intermediates on their folding pathways. In this Opinion article, we suggest that the reason for this impasse is the over-reliance on current physical models of protein folding that are based on the assumption that proteins are able to fold spontaneously without assistance. These models arose from studies conducted in vitro on a biased sample of smaller, easier-to-isolate proteins, whose native structures appear to be thermodynamically stable. Meanwhile, the vast empirical data on the majority of larger proteins suggests that once these proteins are completely denatured in vitro, they cannot fold into native conformations without assistance. Moreover, they tend to lose their native conformations spontaneously and irreversibly in vitro, and therefore such conformations must be metastable. We propose a model of protein folding that is based on the notion that the folding of all proteins in the cell is mediated by the actions of the "protein folding machine" that includes the ribosome, various chaperones, and other components involved in co-translational or post-translational formation, maintenance and repair of protein native conformations in vivo. The most important and universal component of the protein folding machine consists of the ribosome in complex with the welcoming committee chaperones. The concerted actions of molecular machinery in the ribosome peptidyl transferase center, in the exit tunnel, and at the surface of the ribosome result in the application of mechanical and other forces to the nascent peptide, reducing its conformational entropy and possibly creating strain in the peptide backbone. The resulting high-energy conformation of the nascent peptide allows it to fold very fast and to overcome high kinetic barriers along the folding pathway. The early folding intermediates in vivo are stabilized by interactions with the ribosome and welcoming committee chaperones and would not be able to exist in vitro in the absence of such cellular components. In vitro experiments that unfold proteins by heat or chemical treatment produce denaturation ensembles that are very different from folding intermediates in vivo and therefore have very limited use in reconstructing the in vivo folding pathways. We conclude that computational modeling of protein folding should deemphasize the notion of unassisted thermodynamically controlled folding, and should focus instead on the step-by-step reverse engineering of the folding process as it actually occurs in vivo. REVIEWERS This article was reviewed by Eugene Koonin and Frank Eisenhaber.

中文翻译：

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模拟体内蛋白质折叠。

半个世纪以来，在体外研究蛋白质折叠并在计算机上对其建模的研究并没有为我们提供可靠的计算方法来预测从头蛋白质的天然构象，更不用说确定其折叠途径中的中间体了。在此“意见”文章中，我们建议造成这种僵局的原因是过度依赖当前的蛋白质折叠物理模型，该模型基于以下假设：蛋白质能够在没有帮助的情况下自发折叠。这些模型来自于对较小的，更易于分离的蛋白质的偏向样本进行的体外研究，这些蛋白质的天然结构似乎是热力学稳定的。同时，有关大多数较大蛋白的大量经验数据表明，一旦这些蛋白在体外完全变性，他们无法在没有帮助的情况下折叠成天然构象。而且，它们倾向于在体外自发且不可逆地丧失其天然构象，因此这些构象必须是亚稳的。我们提出了一种蛋白质折叠模型，该模型基于以下概念：细胞中所有蛋白质的折叠是由“蛋白质折叠机”的作用介导的，“蛋白质折叠机”包括核糖体，各种分子伴侣和参与共翻译的其他成分或翻译后体内蛋白质天然构象的形成，维持和修复。蛋白质折叠机最重要，最通用的组件是核糖体，与欢迎委员会的陪伴分子复合在一起。分子机制在核糖体肽基转移酶中心，出口通道中的协同作用，并且在核糖体表面导致对新生肽施加机械力和其他力，从而降低了其构象熵，并可能在肽主链中产生应变。新生肽的高能构象使其可以非常快速地折叠，并克服了折叠路径上的高动力学障碍。体内的早期折叠中间体通过与核糖体和欢迎委员会伴侣的相互作用而稳定，并且在缺少这种细胞成分的情况下将无法在体外存在。通过热处理或化学处理使蛋白质展开的体外实验产生的变性集合体与体内折叠中间体非常不同，因此在重建体内折叠途径中的用途非常有限。我们得出的结论是，蛋白质折叠的计算模型应不再强调无辅助热力学控制的折叠的概念，而应着眼于实际上在体内发生的折叠过程的逐步反向工程。审阅者本文由Eugene Koonin和Frank Eisenhaber审阅。