A fluorescent multi-domain protein reveals the unfolding mechanism of Hsp70.
A fluorescent multi-domain protein reveals the unfolding mechanism of Hsp70.
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DOI：
10.1038/s41589-022-01162-9
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发表时间：
2023-02
影响因子：
14.8
通讯作者：
Goloubinoff, Pierre
中科院分区：
文献类型：
--
作者：
Tiwari, Satyam;Fauvet, Bruno;Assenza, Salvatore;De Los Rios, Paolo;Goloubinoff, Pierre
由于缺乏对聚集体的全面表征，对Hsp70伴侣保护细胞免受蛋白质聚集的机制的详细理解受到阻碍，这些聚集体通常是异质的。在这里，我们设计了一个报告分子伴侣底物MLucV，由一个应力不稳定的荧光素酶组成，两侧是抗应力的荧光结构域，在变性后形成一个离散的小聚集体。结合Förster共振能量转移和酶活性测量，提供了聚集，未折叠，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催化的稳定预聚集底物的分解和展开，这将MLucV拉伸到简单展开的构象之外，然后是天然的再折叠。Hsp70在atp驱动下对MLucV聚集体的展开和再折叠作用可能会在高变性温度下积累原生MLucV物种，这对原生状态非常不利。这些结果明确地排除了Hsp70将稳定聚集体转化为亚稳态天然蛋白的非平衡机制中的结合和阻止聚集。Tiwari等人通过设计一种具有良好表征的非天然集合的荧光蛋白，该荧光蛋白捕获了Hsp70/DnaJ/NEF系统对底物的结构调制，从而解决了Hsp70解聚展开机制的各个步骤。
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. By designing a fluorescent protein with a well characterized non-native ensemble, which captures the structural modulation of a substrate by the Hsp70/DnaJ/NEF system, Tiwari et al. resolved the individual steps of the disaggregation unfolding mechanism of Hsp70.
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影响因子：
7.7
作者：
De Los Rios P;Barducci A
通讯作者：
Barducci A
DOI：
10.1073/pnas.0903503106
发表时间：
2009-05-26
影响因子：
11.1
作者：
Bertelsen, Eric B.;Chang, Lyra;Zuiderweg, Erik R. P.
通讯作者：
Zuiderweg, Erik R. P.
影响因子：
7.7
作者：
Assenza, Salvatore;Sassi, Alberto Stefano;Barducci, Alessandro
通讯作者：
Barducci, Alessandro
影响因子：
16
作者：
Kityk, Roman;Kopp, Juergen;Mayer, Matthias P.
通讯作者：
Mayer, Matthias P.
影响因子：
11.4
作者：
Kmiecik, Szymon W.;Le Breton, Laura;Mayer, Matthias P.
通讯作者：
Mayer, Matthias P.