The foundational paradigm of protein folding is that the primary sequence of a protein contains all the information needed for it to adopt a specific native structure. This remarkable property is explained by Anfinsen's thermodynamic hypothesis, which asserts that because native states minimize the Gibbs free energy of a protein molecule under physiological conditions, proteins can reliably navigate to their native structures and remain in those states by ergodically sampling their free energy landscapes. Most experiments of protein folding -- conducted on purified, small, single-domain soluble proteins -- follow the proportion of protein molecules that are folded as a function of time, temperature, denaturant concentration, or sequence, and have yielded immense insight into the molecular determinants that underpin stable globular folds. However, our reliance on the thermodynamic hypothesis as a ground truth to interpret these experiments has limited our ability to study the folding of complex proteins or to consider alternative non-thermodynamic scenarios. Here, we introduce an experimental approach to probe protein refolding for whole proteomes. We accomplish this by first unfolding and refolding E. coli lysates, and then interrogating the resulting protein structures using a permissive protease that preferentially cleaves at flexible regions. Using mass spectrometry, we analyze the digestion patterns to globally assess structural differences between native and "refolded" proteins. These studies reveal that following denaturation, many proteins are incapable of navigating back to their native structures. Our results signal a pervasive role for co-translational folding in shaping protein biogenesis, and suggest that the apparent stability of many native states derive from kinetic persistence rather than thermodynamic stability.

中文翻译：

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不可折叠性遍及整个大肠杆菌蛋白质组

蛋白质折叠的基本范例是蛋白质的一级序列包含其采用特定天然结构所需的所有信息。Anfinsen的热力学假设解释了这种非凡的特性，该假设认为，由于天然状态在生理条件下可将蛋白质分子的吉布斯自由能降至最低，因此蛋白质可以可靠地导航至其天然结构，并通过对它们的自由能态图进行遍历采样来保留在这些状态中。大多数蛋白质折叠实验-是在纯化的，小的单域可溶性蛋白质上进行的-随时间，温度，变性剂浓度或序列的变化而折叠蛋白质分子的比例，并对蛋白质折叠产生了巨大的洞察力。稳定球状褶皱的分子决定簇。但是，我们依靠热力学假设作为解释这些实验的基本事实，限制了我们研究复杂蛋白质折叠或考虑其他非热力学情况的能力。在这里，我们介绍了一种实验方法来探测整个蛋白质组的蛋白质重折叠。我们首先通过展开和重新折叠大肠杆菌裂解物，然后使用优先在柔性区域切割的宽容蛋白酶来查询所得的蛋白质结构，从而实现这一目标。使用质谱法，我们分析了消化模式，以全面评估天然和“折叠”蛋白之间的结构差异。这些研究表明，变性后，许多蛋白质无法导航回其天然结构。