Can microsecond domain motion affect enzymatic activity? A single-molecule FRET study

微秒域运动会影响酶活性吗？

• 批准号: 490757872
• 负责人: Dr. David Scheerer
• 金额: --
• 依托单位: Department of Chemical and Biological Physics
• 依托单位国家: 德国
• 项目类别: WBP Fellowship
• 财政年份: 2021
• 资助国家: 德国
• 起止时间: 2020-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/490757872?language=en
• 关键词: microsecond; domain; motion; affect; enzymatic

Proteins are involved in a broad spectrum of functions, acting as enzymes, signal transductors and more. Many of them have evolved to harness largescale motions of domains and subunits to promote their functionality. Studying the structural dynamics of protein machines is essential for deciphering how they function and in what way they are regulated. The knowledge acquired through such studies is not only very relevant for engineering new proteins, it can also open unprecedented opportunities for the design of drugs.The accurate relation between conformational dynamics and the chemical steps of enzymatic catalysis has been debated extensively. In various enzymes, the active site is situated in a cleft between two domains, and structural rearrangements close the domains over the bound substrate. While current experimental techniques, from x-ray crystallography to cryo-electron microscopy, can readily observe the end points of such motions, measuring the associated time scales is often challenging. Single-molecule Förster resonance energy transfer (smFRET) spectroscopy can measure large-scale motions in real time, with the great advantage that multiple time scales can be covered. Combined with H2MM, a photon-by-photon hidden Markov model analysis, smFRET studies on the enzyme adenylate kinase (AK) have recently revealed very fast domain motions, two orders of magnitude faster than the turnover of the enzyme. AK may use numerous cycles of conformational rearrangement in order to find a relative orientation of its substrates that is optimal for their chemical reaction. Preliminary studies on the effect of urea on the substrate inhibition of AK are in support of this idea, indicating that a delicate balance between domain opening and closing is important for efficient turnover.However, it is not clear yet whether this behavior is specific to AK or applies to a multitude of enzymes, which requires expanding our studies to other proteins. In the enzyme phosphoglycerate kinase (PGK) the catalytic reaction is accompanied by a large-scale hinge-bending motion, rendering it a good target for this evaluation. We will ask: How fast are the motions? What is the relation between catalytic reaction and domain closure? Can the dynamics be described as jumps between two states or is a more complex molecular model required? Are motions within a domain also necessary in order to reach a fully closed state? The smFRET experiments we propose can provide a major contribution to clarify these issues. We aim to see a definitive correlation between the motions of the protein and its activity. Overall, we plan to not only characterize fast, sub-millisecond domain motions in PGK, but also establish a conclusive role for these motions in enzyme catalysis. This has the potential to modify our picture of the relation of dynamics and function dramatically.

蛋白质参与了广泛的功能，作为酶，信号转导和更多。它们中的许多已经进化到利用域和亚基的大规模运动来促进它们的功能。研究蛋白质机器的结构动力学对于破译它们如何发挥作用以及它们以何种方式受到调节至关重要。通过这些研究获得的知识不仅与工程新蛋白质非常相关，而且还可以为药物设计提供前所未有的机会。构象动力学和酶催化化学步骤之间的准确关系一直受到广泛的争论。在各种酶中，活性位点位于两个结构域之间的裂缝中，结构重排使结合底物上的结构域闭合。虽然目前的实验技术，从X射线晶体学到低温电子显微镜，可以很容易地观察到这种运动的终点，但测量相关的时间尺度往往具有挑战性。单分子Förster共振能量转移（smFRET）光谱可以在真实的时间内测量大尺度运动，具有可以覆盖多个时间尺度的巨大优势。结合H2MM，一种逐光子隐马尔可夫模型分析，对腺苷酸激酶（AK）的smFRET研究最近揭示了非常快的域运动，比酶的周转快两个数量级。AK可以使用许多构象重排的循环，以便找到其底物的相对取向，这对于它们的化学反应是最佳的。关于尿素对AK底物抑制作用的初步研究支持了这一观点，表明结构域开放和关闭之间的微妙平衡对于有效的周转是重要的，然而，目前还不清楚这种行为是AK特有的还是适用于多种酶，这需要将我们的研究扩展到其他蛋白质。在酶磷酸甘油酸激酶（PGK）的催化反应是伴随着一个大规模的铰链弯曲运动，使其成为一个很好的目标，这种评估。我们会问：运动有多快？催化反应和畴闭合之间的关系是什么？动力学可以被描述为两个状态之间的跳跃，还是需要一个更复杂的分子模型？为了达到完全闭合的状态，域内的运动也是必要的吗？我们提出的smFRET实验可以为澄清这些问题做出重大贡献。我们的目标是看到蛋白质的运动与其活性之间的明确相关性。 总的来说，我们计划不仅表征快速，亚毫秒的域运动在PGK，但也建立了决定性的作用，这些运动在酶催化。这有可能极大地改变我们对动力学和功能关系的看法。