通过单分子光谱探究变性态特性在伴侣蛋白作用中的角色。
Role of denatured-state properties in chaperonin action probed by single-molecule spectroscopy.
作者信息
Hofmann Hagen, Hillger Frank, Delley Cyrille, Hoffmann Armin, Pfeil Shawn H, Nettels Daniel, Lipman Everett A, Schuler Benjamin
机构信息
Department of Biochemistry, University of Zurich, Zurich, Switzerland.
Department of Biochemistry, University of Zurich, Zurich, Switzerland.
出版信息
Biophys J. 2014 Dec 16;107(12):2891-2902. doi: 10.1016/j.bpj.2014.11.002.
The bacterial chaperonin GroEL/GroES assists folding of a broad spectrum of denatured and misfolded proteins. Here, we explore the limits of this remarkable promiscuity by mapping two denatured proteins with very different conformational properties, rhodanese and cyclophilin A, during binding and encapsulation by GroEL/GroES with single-molecule spectroscopy, microfluidic mixing, and ensemble kinetics. We find that both proteins bind to GroEL with high affinity in a reaction involving substantial conformational adaptation. However, whereas the compact denatured state of rhodanese is encapsulated efficiently upon addition of GroES and ATP, the more expanded and unstructured denatured cyclophilin A is not encapsulated but is expelled into solution. The origin of this surprising disparity is the weaker interactions of cyclophilin A with a transiently formed GroEL-GroES complex, which may serve as a crucial checkpoint for substrate discrimination.
细菌伴侣蛋白GroEL/GroES协助多种变性和错误折叠的蛋白质进行折叠。在此，我们通过单分子光谱、微流控混合和整体动力学，在GroEL/GroES结合和包裹具有非常不同构象性质的两种变性蛋白——硫氰酸酶和亲环蛋白A时，探究这种显著的通用性的限度。我们发现，这两种蛋白质在涉及大量构象适应的反应中都以高亲和力与GroEL结合。然而，虽然硫氰酸酶的紧密变性状态在添加GroES和ATP后能被有效包裹，但更伸展且无结构的变性亲环蛋白A却未被包裹而是被排到溶液中。这种惊人差异的根源是亲环蛋白A与瞬时形成的GroEL-GroES复合物之间的相互作用较弱，这可能是底物识别的关键检查点。
Zotero 插件