Turning symmetric protein scaffolds into robust enablers of structural biology

将对称蛋白质支架转变为结构生物学的强大推动者

• 批准号: BB/T003677/1
• 负责人: Frank Von Delft
• 金额: $ 145.34万
• 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: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FT003677%2F1
• 关键词: Turning; symmetric; protein; scaffolds; into

Images that show relevant biological molecules, their fine details and their interactions, help biochemical and pharmaceutical researchers in their experimental programmes. Structural knowledge of biological molecules has historically transformed our view of what they do and how they do it. Today, such knowledge is increasingly being used to drive the discovery of drugs and vaccines to combat diseases that range from bacterial infection to cancer. Two experimental techniques are key to producing these images. Macromolecular X-ray crystallography (MX) is the most mature method for determining structures of the molecules (usually proteins) that govern the operation of biological systems. However, as recognized by the award of the 2017 Nobel Prize in Chemistry, electron microscopy (EM) has recently come of age as a viable alternative for atomic resolution visualization of large macromolecules. Despite significant technical advances in recent years for both MX and EM, these two techniques do not yet provide a complete answer to observing all biological molecules in a timely fashion. EM is typically only used to study relatively large biological molecules and complexes. X-ray crystallography is less demanding in respect of size, but, requires the protein of interest to form crystals - billions of identical copies arrayed in a near-faultless three-dimensional grid. This can only be accomplished by trial and error, by testing a protein in thousands of different solutions to see if it can be persuaded to crystallise. Alas, this fails more often than not, and for many of the most therapeutically interesting, it is difficult if not impossible.Our approach to addressing this issue is to prepare nano-sized 3D scaffolds ("crysalins") on to which a protein of choice can be attached in a crystalline array. Whilst we have successfully produced the nano-scaffolds and attached examplar targets onto them, we remain one step away from this becoming a universal platform for structure determination. Currently the target proteins are attached only loosely to the scaffold and, because X-rays show you an average picture of the repeating unit of the array, any looseness "smears out" the image of the target protein. This programme of work will firm up the connection to ensure that each target molecule is attached to the scaffold in exactly the same position and orientation.An added benefit of developing rigid target-scaffold connections is that the building blocks that make up our scaffolds are large and symmetric and therefore perfectly suited to imaging on an electron microscope. Even small targets (of a size usually inaccessible to EM) that are rigidly and uniformly attached to such building blocks have been shown to be straightforwardly imaged by EM. We are therefore producing a building block that can either be used in isolation to facilitate electron microscopy or assembled into a lattice to enable crystallography.An area in which MX still reigns supreme is high-throughput structure determination, especially where one is looking at a single protein target binding to a number of different small chemicals. If suitable crystals of a protein are available, they can be soaked in solutions of these chemicals and imaged. Indeed, one can screen a large library of chemicals and trawl through the structural images to find candidates that might be refined as therapeutic drugs. The whole process of refinement can be done rationally, seeing how variants of the original chemical bind and how their shape and properties might be changed in order to improve them to enhance their effectiveness. This process is known as structure-based ligand design, SBLD. We will demonstrate the potential of crysalin technology for SBLD in conjuction with the "XChem" chemical screening platform and apply it to proteins that are potential therapeutic targets under study in the laboratories of the PIs.

显示相关生物分子及其精细细节和相互作用的图像有助于生物化学和制药研究人员进行实验计划。生物分子的结构知识历史性地改变了我们对它们做什么以及如何做的看法。今天，这些知识正越来越多地用于推动药物和疫苗的发现，以对抗从细菌感染到癌症的各种疾病。两种实验技术是产生这些图像的关键。大分子x射线晶体学（MX）是测定控制生物系统运行的分子（通常是蛋白质）结构的最成熟的方法。然而，正如2017年诺贝尔化学奖所承认的那样，电子显微镜（EM）最近已经成为大分子原子分辨率可视化的可行替代方案。尽管近年来MX和EM技术取得了重大进展，但这两种技术还不能提供及时观察所有生物分子的完整答案。EM通常只用于研究相对较大的生物分子和复合物。x射线晶体学对大小的要求较低，但它需要感兴趣的蛋白质形成晶体——数十亿相同的副本排列在近乎完美的三维网格中。这只能通过反复试验来完成，通过在数千种不同的溶液中测试一种蛋白质，看看它是否能被说服结晶。唉，这种方法往往会失败，而且对于许多最具治疗意义的人来说，即使不是不可能，也是很困难的。我们解决这个问题的方法是制备纳米尺寸的3D支架（“晶体蛋白”），选择的蛋白质可以在其上以晶体阵列的形式附着。虽然我们已经成功地生产了纳米支架，并将示例靶标附着在其上，但我们离成为结构测定的通用平台还有一步之遥。目前，目标蛋白质只是松散地附着在支架上，由于x射线显示的是阵列重复单元的平均图像，任何松动都会“涂抹”目标蛋白质的图像。这项工作将巩固连接，以确保每个目标分子以完全相同的位置和方向附着在支架上。开发刚性目标-支架连接的另一个好处是，构成支架的构件是大而对称的，因此非常适合在电子显微镜上成像。即使是很小的目标（通常是EM无法达到的大小），这些目标都是严格且均匀地附着在这样的构建块上，EM已经证明可以直接成像。因此，我们正在生产一种构建块，既可以单独使用以方便电子显微镜，也可以组装成晶格以实现晶体学。MX仍然占据主导地位的一个领域是高通量结构测定，特别是当人们观察与许多不同的小化学物质结合的单个蛋白质目标时。如果有合适的蛋白质晶体，可以将它们浸泡在这些化学物质的溶液中并成像。事实上，人们可以筛选大量的化学物质，并通过结构图像来寻找可能被提炼为治疗药物的候选物质。整个精炼过程可以合理地进行，看看原始化学物质的变体是如何结合的，它们的形状和性质是如何改变的，以改进它们，提高它们的有效性。这个过程被称为基于结构的配体设计（sld）。我们将结合“XChem”化学筛选平台展示水晶素技术在SBLD中的潜力，并将其应用于pi实验室研究的潜在治疗靶点蛋白质。