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Optima Analytical Ultracentrifuge for mechanistic insights into complex protein binding events

Optima 分析超速离心机可深入了解复杂蛋白质结合事件的机制

基本信息

批准号：
BB/W019841/1
负责人：
Thomas Jowitt
金额：
$ 33.27万
依托单位：
University of Manchester
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：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FW019841%2F1
关键词：
Optima Analytical Ultracentrifuge mechanistic insights

项目摘要

Protein complexes that are made-up of multiple different proteins and often multiples of the same protein, are inherently difficult to study. Correct assembly of these proteins is essential for our immune response to pathogens, such as viruses, and for our tissues, such as kidneys to function correctly. Therefore, knowledge of the mechanism behind how these complexes are formed is fundamental to our understanding of key biological processes and why, when they don't form properly, this can lead to disease. The AUC is one of the only methods of studying the stoichiometry of multi-component complexes and the data that is generated is highly complementary to data gained from single-molecule electron microscopy and crystallography, which together allow us to interrogate the mechanism involved in complex formation at the molecular level and is vital to our ongoing research into fundamental biological mechanisms, drug discovery and synthetic biology.The proposed equipment is the Optima Analytical Ultracentrifuge by Beckman. An AUC is a high-speed centrifuge with optical devices fitted that allows the user to follow the movement of particles in a centrifugal field, in our case the particles are predominantly protein molecules. The rate of movement of the particles through a liquid (sedimentation) is related to their mass, conformation and effects of the solute. Over the last few decades, AUC instruments have evolved to house different detection systems and improved data analysis has allowed separation of discrete species within a complex mixture. However, in older instruments, it is not possible to identify the different sedimenting species observed in a complex sample. For example, we cannot tell if there are dimers of one protein interacting with monomers of another or vice versa therefore severely limiting usability. The new Optima AUC has far greater signal-to-noise and faster scan-rates meaning multiple wavelengths can be scanned at once. For the first time, this allows the sedimentation of multiple species to be monitored in the same cell which opens up exciting possibilities for the investigation of protein complex formation.The new AUC will allow us to monitor multiple absorbance profiles instantaneously, providing an unparalleled insight into complex assembly processes that we cannot currently do.Information gained from the AUC has also been extremely beneficial to the university and the pharmaceutical-industry in understanding conformational changes induced by drug target interactions (see letter of support). It is in fact the only method that can separate differences in conformation and oligomerisation induced by drug binding. This application aims to provide continued access to analytical ultracentrifugation for the local and national pharmaceutical sector and for the N8 University partnership, for which this will be the only Optima AUC instrument. There are only 2 others currently in UK universities, with one at Harwell available for external use. The new Optima AUC will provide additional exciting capabilities to projects from the main users, who are co-applicants, and include the analysis of extracellular matrix proteins involved in cell signalling, tissue strength and inflammation; determining the structures of membrane proteins responsible for multidrug resistance and kidney function; investigation of enzyme mechanism, biocatalysis and protein formulation.The instrument will also form part of the BACF training workshops and will be an integral part of hydrodynamics training for the next generation of biochemical scientists.

由多个不同蛋白质组成的蛋白质复合体，通常是同一蛋白质的多个，本质上是很难研究的。这些蛋白质的正确组装对于我们对病原体(如病毒)的免疫反应以及我们的组织(如肾脏)的正常运作至关重要。因此，了解这些复合体是如何形成的机制对于我们理解关键的生物过程以及为什么当它们没有正确形成时会导致疾病是基本的。AUC是研究多组分络合物化学计量比的唯一方法之一，所产生的数据与单分子电子显微镜和结晶学获得的数据高度互补，这些数据使我们能够在分子水平上询问络合物形成的机理，对于我们正在进行的基本生物学机制、药物发现和合成生物学的研究至关重要。建议使用的设备是Beckman的Optima分析超速离心机。AUC是一种装有光学设备的高速离心机，允许用户跟踪粒子在离心场中的运动，在我们的情况下，粒子主要是蛋白质分子。粒子在液体中的运动速度与它们的质量、构象和溶质的作用有关。在过去的几十年里，AUC仪器已经发展成容纳不同的检测系统，并且改进的数据分析允许分离复杂混合物中的离散物种。然而，在较旧的仪器中，不可能识别在复杂样品中观察到的不同沉积物种。例如，我们无法判断是否存在一种蛋白质的二聚体与另一种蛋白质的单体相互作用，反之亦然，因此严重限制了可用性。新的Optima AUC具有更高的信噪比和更快的扫描速率，这意味着可以同时扫描多个波长。这是第一次允许在同一个细胞中监测多种物种的沉积，这为研究蛋白质复合体的形成打开了令人兴奋的可能性。新的AUC将使我们能够同时监测多个吸收曲线，提供我们目前无法做到的对复杂组装过程的无与伦比的洞察。从AUC获得的信息也对大学和制药行业了解药物靶标相互作用引起的构象变化非常有益(见支持函)。事实上，这是唯一可以区分药物结合引起的构象差异和寡聚的方法。这项申请旨在为地方和国家制药部门以及N8大学伙伴关系提供持续的分析超速离心法，这将是唯一的最佳AUC仪器。目前只有两所英国大学在使用，其中一所在哈威尔大学，可供外部使用。新的Optima AUC将为共同申请的主要用户的项目提供更多令人兴奋的能力，包括分析细胞信号、组织强度和炎症所涉及的细胞外基质蛋白质；确定与多药耐药性和肾脏功能有关的膜蛋白质的结构；研究酶机制、生物催化和蛋白质配方。该仪器还将成为BACF培训研讨会的一部分，并将成为下一代生化科学家流体力学培训的组成部分。