Detailed understanding of the mechanism by which Hsp70 chaperones protect cells against protein aggregation is hampered by the lack of a comprehensive characterization of the aggregates, which are typically heterogeneous. Here we designed a reporter chaperone substrate, MLucV, composed of a stress-labile luciferase flanked by stress-resistant fluorescent domains, which upon denaturation formed a discrete population of small aggregates. Combining Förster resonance energy transfer and enzymatic activity measurements provided unprecedented details on the aggregated, unfolded, Hsp70-bound and native MLucV conformations. The Hsp70 mechanism first involved ATP-fueled disaggregation and unfolding of the stable pre-aggregated substrate, which stretched MLucV beyond simply unfolded conformations, followed by native refolding. The ATP-fueled unfolding and refolding action of Hsp70 on MLucV aggregates could accumulate native MLucV species under elevated denaturing temperatures highly adverse to the native state. These results unambiguously exclude binding and preventing of aggregation from the non-equilibrium mechanism by which Hsp70 converts stable aggregates into metastable native proteins.

对 Hsp70 伴侣保护细胞免受蛋白质聚集的机制的详细了解因缺乏对聚集体的全面表征而受到阻碍，这些聚集体通常是异质的。在这里，我们设计了一种报告分子伴侣底物 MLucV，它由两侧是抗压荧光结构域的应力不稳定荧光素酶组成，变性后形成离散的小聚集体群。结合 Förster 共振能量转移和酶活性测量，提供了关于聚集、展开、Hsp70 结合和天然 MLucV 构象的前所未有的细节。Hsp70 机制首先涉及 ATP 驱动的稳定预聚集底物的解聚和展开，这将 MLucV 拉伸到简单展开的构象之外，然后是天然重新折叠。Hsp70 对 MLucV 聚集体的 ATP 驱动的展开和重折叠作用可以在升高的变性温度下积累天然 MLucV 物种，这对天然状态非常不利。这些结果明确排除了 Hsp70 将稳定聚集体转化为亚稳态天然蛋白质的非平衡机制中的结合和防止聚集。