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Breaking the Cage: Transformative Time-resolved Crystallography using Fixed Targets at Synchrotrons and XFELs

打破牢笼：在同步加速器和 XFEL 上使用固定目标的变革性时间分辨晶体学

基本信息

批准号：
BB/W001950/1
负责人：
Jonathan Worrall
金额：
$ 56.85万
依托单位：
University of Essex
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2022
资助国家：
英国
起止时间：
2022 至无数据
项目状态：
未结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FW001950%2F1
关键词：
Breaking Cage Transformative Time resolved

项目摘要

Efforts to understand how enzyme catalysis and protein-ligand binding work (or how they fail to work properly) is a vital research field in the life sciences. The knowledge gained has significance for the development of efficient biosynthetic materials, for applications in the biochemical, biotechnology and industrial sectors and in developing new medicines. Obtaining the structures of enzymes is an essential part of this effort. X-ray crystallography is a technique that reveals the individual atoms that make up an enzyme molecule, showing how they are joined to each other to form larger 3D structures. These structures and how they change over time as an enzyme performs its work are key factors for our understanding of enzyme function. This 'time-resolved' aspect is analogous to moving from a single photograph to a movie, and is tremendously powerful but also very challenging to achieve. To uncover this detailed 3D arrangement of atoms within an enzyme, powerful X-ray sources, called synchrotron storage rings and X-ray free electron lasers (XFELs), are used to visualise crystallised versions of the enzyme, using micron sized X-ray beams. The structure of the enzyme can then be deduced from the way that the X-rays are 'diffracted' as they pass through the crystal. The crystals that are grown in the laboratory for these experiments, including many important pharmaceutical targets for therapies, are very often only available as tiny, micron-sized (one-thousandth of a millimetre) 'microcrystals'. We will use silicon 'chips' for efficient delivery to the X-ray beam, for optimum high-throughput and high 'hit rate' (a 'hit' is when the micron size X-ray beam is diffracted by a microcrystal). A chip is a device containing thousands of shaped wells each of which can trap a microcrystal. Each chip can hold up to 25,600 microcrystals and each well, with its trapped microcrystal, is exposed in turn to the X-rays beam at the selected experimental facility used (the Diamond synchrotron in the UK or SACLA in Japan). The different facilities used have very different properties so can be used to provide complementary information by for example probing different timescales.The X-ray diffraction patterns gathered from all the microcrystal hits are combined and analysed to create a 3D model (structure) of the enzyme. Using chips for sample delivery we can obtain a complete 3D structure of an enzyme in less than one hour, which is a very efficient use of these expensive X-ray facilities. We will first study enzymes in their 'resting' states before they undergo catalysis (do their work), giving us the initial structures of the enzymes. We will use oxygen and nitric oxide and photocages to initiate catalysis or ligand binding in microcrystals. Photocages are compounds that contain a trapped molecule of interest, which is held securely and only released by a brief flash of a laser beam. By collecting diffraction patterns from the microcrystals at varying time delays after the laser flash we can capture time dependent changes and build up an accurate molecular movie of the enzyme in action. Crucially, we are able to produce these structures at room temperature, close to the conditions under which enzymes function in nature. This allows their dynamic movements related to their function to be followed much more accurately compared to the cryogenic conditions (liquid nitrogen temperature, approximately -173 oC) at which crystal structures are typically produced. Our approach allows us to follow reactions over a wide timescale from microseconds to seconds or minutes, building up a complete picture of catalysis.

努力了解酶催化和蛋白质-配体结合如何工作（或它们如何不能正常工作）是生命科学中一个重要的研究领域。所获得的知识对于开发有效的生物合成材料，应用于生物化学，生物技术和工业部门以及开发新药具有重要意义。获得酶的结构是这项工作的重要组成部分。X射线晶体学是一种技术，它揭示了构成酶分子的单个原子，显示了它们如何相互连接以形成更大的3D结构。这些结构以及它们在酶执行其工作时如何随时间变化是我们理解酶功能的关键因素。这种“时间分辨”的方式类似于从一张照片到一部电影，非常强大，但也非常具有挑战性。为了揭示酶中原子的这种详细的3D排列，使用称为同步加速器存储环和X射线自由电子激光器（XFEL）的强大X射线源，使用微米大小的X射线束来可视化酶的结晶版本。然后，可以根据X射线穿过晶体时“衍射”的方式推导出酶的结构。在实验室中为这些实验生长的晶体，包括许多重要的药物治疗靶点，通常只能作为微小的微米级（千分之一毫米）“微晶”。我们将使用硅“芯片”来有效地传递X射线束，以获得最佳的高吞吐量和高“命中率”（“命中率”是指微米尺寸的X射线束被微晶衍射时）。芯片是一种包含数千个成型威尔斯的装置，每个井都可以捕获微晶。每个芯片可以容纳多达25，600个微晶，每个阱及其捕获的微晶依次暴露于所使用的选定实验设施（英国的钻石同步加速器或日本的SACLA）的X射线束。所使用的不同设备具有非常不同的特性，因此可以通过例如探测不同的时间尺度来提供互补信息。从所有微晶命中收集的X射线衍射图案被组合和分析，以创建酶的3D模型（结构）。使用芯片进行样品传输，我们可以在不到一个小时的时间内获得酶的完整3D结构，这是对这些昂贵的X射线设备的非常有效的使用。我们将首先研究酶在进行催化之前的“静止”状态，为我们提供酶的初始结构。我们将利用氧、一氧化氮和光笼来引发微晶中的催化或配体结合。光笼是一种包含感兴趣的捕获分子的化合物，该分子被牢固地固定并且仅通过激光束的短暂闪光释放。通过在激光闪光后的不同时间延迟收集来自微晶的衍射图案，我们可以捕获时间依赖性变化并建立起酶作用的精确分子电影。最重要的是，我们能够在室温下产生这些结构，接近酶在自然界中发挥作用的条件。这使得与晶体结构通常产生的低温条件（液氮温度，约-173 ° C）相比，可以更准确地跟踪与其功能相关的动态运动。我们的方法使我们能够在从微秒到秒或分钟的广泛时间尺度上跟踪反应，从而建立起催化的完整画面。