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Single protein crystal spectroscopy and crystallography of hydrogenase under electrochemical control

电化学控制下氢化酶的单蛋白晶体光谱和晶体学

基本信息

批准号：
BB/R018413/1
负责人：
Kylie Vincent
金额：
$ 82.13万
依托单位：
University of Oxford
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2018
资助国家：
英国
起止时间：
2018 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FR018413%2F1
关键词：
Single protein crystal spectroscopy crystallography

项目摘要

Proteins are the nanoscale 'machines' that control almost all processes in cells. Importantly they are responsible for speeding up (catalysing) chemical reactions that make the essential molecules for life and release or store energy. Understanding the structures of individual proteins at the atomic level has been absolutely key in building up our understanding of how they contribute to the function of cells. In particular, the technique of X-ray crystallography has been extremely valuable in providing 'snapshot' images of many different proteins at the level of individual atoms. In this approach, crystals of the protein are prepared, and are probed using a high energy X-ray source which diffracts offindividual atoms in the crystal, giving a pattern that reveals the positions of atoms in the protein. A series of these 'snapshots' is needed to understand each step in how a protein works, and it is often difficult to trap proteins in specific states relevant to their function in order to obtain a complete set of 'snapshots'. This is particularly true for a group of 'redox' proteins which catalyse chemical reactions involving the transfer of electrons - oxidation and reduction reactions. A further challenge arises because these redox proteins often incorporate metal atoms that are susceptible to damage by X-rays during the data collection for crystal structure determination. It is very timely that we find new tools for studying these sort of proteins, because they catalyse many chemical reactions which are relevant to solving big global challenges, including how to use hydrogen as a sustainable fuel, how to capture the greenhouse gas carbon dioxide and turn it into useful chemicals, and how to efficiently produce the fertilisers needed to sustain the world's growing population. In this project, we demonstrate a completely new approach to controlling and verifying the state of redox proteins that will allow crystal structure snapshots to be produced for many more of the important functional states of these redox proteins. Our focus is a protein called hydrogenase which allows microbes to live on hydrogen gas as their energy source. In earlier preliminary work, we have shown that we can use electrodes to control a single crystal of hydrogenase to generate uniform states relevant to its function. This provides an unprecedented way to get proteins in single crystals into specific states ready to record X-ray crystallographic 'snapshots'. At the same time, we make use of imaging using infrared light with a special infrared microscope to confirm the state of the protein in the crystal. During the project, we will show that we can prepare specific states of protein crystals in this way and then record their X-ray structures to yield snapshots of previously unseen states of the protein. This will yield new information on how proteins function as efficient catalysts for the important reactions mentioned above. We will also use the infrared imaging approach to check the crystals after exposure to X-rays to make sure that the state of the protein in the crystal has not been damaged during X-ray crystallographic data collection. This approach will lead to much more reliable snapshots of redox proteins.The project thus represents a step change for structural biology of redox proteins and understanding the function of proteins which may teach us how to solve important global problems. We would like these tools to become widely available to structural biologists who solve the structures of complicated proteins, and during this project we aim to develop our approaches so that they can be readily implemented.What we learn about the way that hydrogenases work in the course of this project will help to propel biotechnological applications of hydrogenases, and will underpin development of alternative chemical catalysts based on the cheap metals, nickel and iron, found inside the hydrogenases.

蛋白质是控制细胞中几乎所有过程的纳米级“机器”。重要的是，它们负责加速（催化）化学反应，使生命的基本分子和释放或储存能量。在原子水平上理解单个蛋白质的结构对于我们理解它们如何促进细胞功能至关重要。特别是，X射线晶体学技术在提供许多不同蛋白质在单个原子水平上的“快照”图像方面非常有价值。在这种方法中，制备蛋白质晶体，并使用高能X射线源进行探测，该X射线源衍射晶体中的单个原子，给出揭示蛋白质中原子位置的图案。需要一系列这些“快照”来了解蛋白质如何工作的每一步，并且通常很难将蛋白质捕获在与其功能相关的特定状态下，以获得一组完整的“快照”。这对于一组“氧化还原”蛋白质尤其如此，它们催化涉及电子转移的化学反应-氧化和还原反应。另一个挑战出现了，因为这些氧化还原蛋白质通常含有金属原子，这些金属原子在晶体结构测定的数据收集过程中容易受到X射线的破坏。我们发现研究这类蛋白质的新工具是非常及时的，因为它们催化许多与解决全球重大挑战有关的化学反应，包括如何使用氢作为可持续燃料，如何捕获温室气体二氧化碳并将其转化为有用的化学品，以及如何有效地生产维持世界人口增长所需的肥料。在这个项目中，我们展示了一种全新的方法来控制和验证氧化还原蛋白的状态，这将允许为这些氧化还原蛋白的更多重要功能状态生成晶体结构快照。我们的重点是一种叫做氢化酶的蛋白质，它允许微生物以氢气为能源。在早期的初步工作中，我们已经表明，我们可以使用电极来控制氢化酶的单晶，以产生与其功能相关的均匀状态。这提供了一种前所未有的方法，可以使单晶中的蛋白质进入特定状态，准备记录X射线晶体学“快照”。同时，我们使用特殊的红外显微镜利用红外光成像来确认晶体中蛋白质的状态。在该项目中，我们将证明我们可以以这种方式制备蛋白质晶体的特定状态，然后记录它们的X射线结构，以生成蛋白质以前未见过的状态的快照。这将产生关于蛋白质如何作为上述重要反应的有效催化剂的新信息。我们还将使用红外成像方法检查暴露于X射线后的晶体，以确保晶体中蛋白质的状态在X射线晶体学数据收集期间没有被破坏。这种方法将导致氧化还原蛋白质的更可靠的快照。因此，该项目代表了氧化还原蛋白质的结构生物学和理解蛋白质功能的一个步骤，这可能会教我们如何解决重要的全球性问题。我们希望这些工具能广泛应用于解决复杂蛋白质结构的结构生物学家，在这个项目中，我们的目标是发展我们的方法，以便它们可以容易地实施。我们在这个项目过程中对氢化酶工作方式的了解将有助于推动氢化酶的生物技术应用，并将支持替代化学催化剂的开发，这些催化剂基于氢化酶中发现的廉价金属镍和铁。

项目成果

期刊论文数量（10）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

E. coli Nickel-Iron Hydrogenase 1 Catalyses Non-native Reduction of Flavins: Demonstration for Alkene Hydrogenation by Old Yellow Enzyme Ene-reductases**

大肠杆菌镍铁氢化酶 1 催化黄素的非天然还原：老黄酶烯还原酶对烯烃氢化的演示**

DOI：
10.1002/ange.202101186
发表时间：
2021
期刊：
Angewandte Chemie
影响因子：
0
作者：
Joseph Srinivasan S
通讯作者：
Joseph Srinivasan S

Electrochemical control of [FeFe]-hydrogenase single crystals reveals complex redox populations at the catalytic site.

DOI：
10.1039/d1dt02219a
发表时间：
2021-09-21
期刊：
Dalton transactions (Cambridge, England : 2003)
影响因子：
0
作者：
Morra S;Duan J;Winkler M;Ash PA;Happe T;Vincent KA
通讯作者：
Vincent KA

Electrochemical experiments define potentials associated with binding of substrates and inhibitors to nitrogenase MoFe protein.

DOI：
10.1039/d2fd00170e
发表时间：
2023-07-19
期刊：
FARADAY DISCUSSIONS
影响因子：
3.4
作者：
Chen, Ting;Ash, Philip A.;Seefeldt, Lance C.;Vincent, Kylie A.
通讯作者：
Vincent, Kylie A.

Structural basis for bacterial energy extraction from atmospheric hydrogen.

DOI：
10.1038/s41586-023-05781-7
发表时间：
2023-03
期刊：
NATURE
影响因子：
64.8
作者：
Grinter, Rhys;Kropp, Ashleigh;Venugopal, Hari;Senger, Moritz;Badley, Jack;Cabotaje, Princess R.;Jia, Ruyu;Duan, Zehui;Huang, Ping;Stripp, Sven T.;Barlow, Christopher K.;Belousoff, Matthew;Shafaat, Hannah S.;Cook, Gregory M.;Schittenhelm, Ralf B.;Vincent, Kylie A.;Khalid, Syma;Berggren, Gustav;Greening, Chris
通讯作者：
Greening, Chris

Comprehensive structural, infrared spectroscopic and kinetic investigations of the roles of the active-site arginine in bidirectional hydrogen activation by the [NiFe]-hydrogenase 'Hyd-2' from Escherichia coli.