The proteasome is a key player of regulated protein degradation in all kingdoms of life. Although recent atomic structures have provided snapshots on a number of conformations, data on substrate states and populations during the active degradation process in solution remain scarce. Here, we use time-resolved small-angle neutron scattering of a deuterium-labeled GFPssrA substrate and an unlabeled archaeal PAN-20S system to obtain direct structural information on substrate states during ATP-driven unfolding and subsequent proteolysis in solution. We find that native GFPssrA structures are degraded in a biexponential process, which correlates strongly with ATP hydrolysis, the loss of fluorescence, and the buildup of small oligopeptide products. Our solution structural data support a model in which the substrate is directly translocated from PAN into the 20S proteolytic chamber, after a first, to our knowledge, successful unfolding process that represents a point of no return and thus prevents dissociation of the complex and the release of harmful, aggregation-prone products.

中文翻译：

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通过时间分辨中子散射观察 PAN-20S 蛋白酶体对蛋白质的降解

蛋白酶体是所有生命王国中调节蛋白质降解的关键参与者。尽管最近的原子结构已经提供了许多构象的快照，但溶液中主动降解过程中底物状态和种群的数据仍然很少。在这里，我们使用氘标记的 GFPssrA 底物和未标记的古菌 PAN-20S 系统的时间分辨小角度中子散射，在 ATP 驱动的展开和随后的溶液中蛋白水解过程中获得有关底物状态的直接结构信息。我们发现天然 GFPssrA 结构在双指数过程中降解，这与 ATP 水解、荧光损失和小寡肽产物的积累密切相关。