Optima Analytical Ultracentrifuge for mechanistic insights into complex protein binding events

由多种不同蛋白质组成的蛋白质复合物，通常是同一蛋白质的倍数，本质上很难研究。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）的正常功能至关重要。因此，了解这些复合物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不能正常形成时会导致疾病至关重要。AUC是研究多组分复合物化学计量的唯一方法之一，生成的数据与单分子电子显微镜和晶体学获得的数据高度互补，它们共同使我们能够探究复合物形成所涉及的机制在分子水平上，对于我们正在进行的基础生物学机制研究至关重要，建议的设备是Beckman的Optima Analytical Ultracenthalge。AUC是一种装有光学装置的高速离心机，允许用户跟踪离心场中颗粒的运动，在我们的情况下，颗粒主要是蛋白质分子。粒子在液体中的运动速率（沉降）与它们的质量、构象和溶质的影响有关。在过去的几十年里，AUC仪器已经发展到容纳不同的检测系统，并且改进的数据分析已经允许分离复杂混合物中的离散物质。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类。例如，我们无法判断一种蛋白质的二聚体是否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。这是第一次在同一个池中监测多个物质的沉降，为研究蛋白质复合物的形成开辟了令人兴奋的可能性。新的AUC将使我们能够即时监测多个吸光度曲线，提供了一个无与伦比的洞察复杂的装配过程，我们目前还不能做。从AUC获得的信息也非常有益的大学和在理解药物靶点相互作用引起的构象变化方面，制药工业提供了大量的信息（见支持函）。事实上，这是唯一的方法，可以分开的构象和药物结合诱导的寡聚化的差异。该应用旨在为当地和国家制药部门以及N8大学合作伙伴关系提供持续的分析超浓缩，这将是唯一的Optima AUC仪器。目前英国大学中只有2个，其中一个在Harwell可供外部使用。新的Optima AUC将为主要用户（共同申请人）的项目提供额外的令人兴奋的功能，包括分析参与细胞信号传导、组织强度和炎症的细胞外基质蛋白;确定负责多药耐药性和肾功能的膜蛋白的结构;酶作用机理研究，该仪器也将成为BACF培训讲习班的一部分，并将成为下一个流体力学培训的一个组成部分。一代生化科学家。

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.