PNAS：核磁共振揭开分子伴侣的神秘面纱

PNAS：核磁共振揭开分子伴侣的神秘面纱 2013-06-27 来源：ebiotrade 作者：koo HTMLCONTROL Forms.HTML:Hidden.1 HYPERLINK "javascript:unlogin();" 收藏(2) 日前，美国国立卫生研究院的科学家们将四种核磁共振技术结合起来，首次解析了两个重要蛋白之间的关键相互作用。这项研究展示了分子伴侣与无序蛋白的结合过程，这样的相互作用广泛存在于生物界。分子伴侣能与较小的无序蛋白结合，并帮助它们形成特定的构象，这是保证蛋白正确折叠的重要机制。 研究人员对胶囊状的细菌分子伴侣GroEL，和无序的β-淀粉样蛋白（A-beta）进行研究，A-beta是阿尔茨海默症中的核心蛋白。人们对这两种蛋白都很熟悉，但并不清楚GroEL胶囊是如何开启并捕获目标的。 NMR能够反映蛋白之间的相互作用及其动力学，但单一的NMR往往不能提供全面的信息。为此，NIH的研究人员将四种不同的NMR技术结合起来，成功解析了A-beta与GroEL结合时的结构和动态。 研究人员发现，GroEL只在两个疏水区域与A-beta结合，使较小的A-beta蛋白可以在多种构象间转变。这两个蛋白并没有全程结合在一起，而是在不断的解离和重新结合。事实上，A-beta会时不时地在GroEL的结合腔中跳跃。 研究人员利用四种NMR获得了丰富的信息，包括蛋白结合时的结构、蛋白解离/重新结合的频率，以及蛋白在不同区域的活动。研究人员表示，A-beta在结合时并没有形成特定的结构，而是保持着构象的灵活性。 研究人员指出，GroEL的这种松散方式，使它能够结合多种不同的无序蛋白，同时也节省了能量。如果要在捕获初期强迫蛋白形成特殊构象，就需要更多的能量，而这样做并不划算。 研究显示，GroEL在初始结合后关闭，将目标蛋白完全装入胶囊。此时，分子伴侣才会投入能量，促使蛋白以正确的方式折叠。 对于分子和结构生物学家来说，这种将NMR技术混合使用的方式，可以帮助他们阐明之前难以解析的相互作用。 研究人员总结道：这种方法可以为人们展示，更多大型蛋白机器的作用机制。 原文检索： David S. Libich, Nicolas L. Fawzi, Jinfa Ying and G. Marius Clore. Probing the transient dark state of substrate binding to GroEL by relaxation-based solution NMR. PNAS June 24, 2013; doi:10.1073/pnas.1305715110 Probing the transient dark state of substrate binding to GroEL by relaxation-based solution NMR David S. Libich HYPERLINK "http://www.pnas.org/content/early/2013/06/20/1305715110.abstract" \l "aff-1#aff-1" a,1, Nicolas L. Fawzi HYPERLINK "http://www.pnas.org/content/early/2013/06/20/1305715110.abstract" \l "aff-1#aff-1" a,b,1, Jinfa Ying HYPERLINK "http://www.pnas.org/content/early/2013/06/20/1305715110.abstract" \l "aff-1#aff-1" a, and G. Marius Clore HYPERLINK "http://www.pnas.org/content/early/2013/06/20/1305715110.abstract" \l "aff-1#aff-1" a,2 Author Affiliations aLaboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520; and bDepartment of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, RI 02912 Edited* by George H. Lorimer, University of Maryland, College Park, MD, and approved May 20, 2013 (received for review March 25, 2013) Abstract The mechanism whereby the prototypical chaperonin GroEL performs work on substrate proteins has not yet been fully elucidated, hindered by lack of detailed structural and dynamic information on the bound substrate. Previous investigations have produced conflicting reports on the state of GroEL-bound polypeptides, largely due to the transient and dynamic nature of these complexes. Here, we present a unique approach, based on combined analysis of four complementary relaxation-based NMR experiments, to probe directly the “dark” NMR-invisible state of the model, intrinsically disordered, polypeptide amyloid β (Aβ40) bound to GroEL. The four NMR experiments, lifetime line-broadening, dark-state exchange saturation transfer, relaxation dispersion, and small exchange-induced chemical shifts, are dependent in different ways on the overall exchange rates and populations of the free and bound states of the substrate, as well as on residue-specific dynamics and structure within the bound state as reported by transverse magnetization relaxation rates and backbone chemical shifts, respectively. Global fitting of all the NMR data shows that the complex is transient with a lifetime of <1 ms, that binding involves two predominantly hydrophobic segments corresponding to predicted GroEL consensus binding sequences, and that the structure of the bound polypeptide remains intrinsically and dynamically disordered with minimal changes in secondary structure propensity relative to the free state. Our results establish a unique method to observe NMR-invisible dynamic states of GroEL-bound substrates and to describe at atomic resolution the events between substrate binding and encapsulation that are crucial for understanding the normal and stress-related metabolic function of chaperonins. supramolecular machine; protein–protein interactions;conformational sampling · Footnotes · ↵1D.S.L. and N.L.F. contributed equally to this work. · ↵2To whom correspondence should be addressed. E-mail: mariusc@mail.nih.gov. · Author contributions: D.S.L., N.L.F., J.Y., and G.M.C. designed research, performed research, analyzed data, and wrote the paper. · The authors declare no conflict of interest. · ↵*This Direct Submission article had a prearranged editor. · This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1305715110/-/DCSupplemental. _1433873703.unknown _1433873702.unknown