Optima Analytical Ultracentrifuge for mechanistic insights into complex protein binding events

由多种不同的蛋白质组成的蛋白质复合物，通常是同一蛋白质的倍数，本质上是难以研究的。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，了解这些复合物如何形成背后的机制是我们理解关键生物过程的基础，以及为什么当它们不能正确形成时，就会导致疾病。AUC是研究多组分复合物化学计量学的唯一方法之一，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，这两种方法使我们能够在分子水平上询问复杂形成的机制，对我们正在进行的基础生物学机制，药物发现和合成生物学的研究至关重要。建议的设备是贝克曼公司的Optima分析超离心机。AUC是一种高速离心机，配有光学装置，允许用户在离心场中跟踪粒子的运动，在我们的例子中，粒子主要是蛋白质分子。颗粒通过液体（沉降）的运动速率与它们的质量、构象和溶质的影响有关。在过去的几十年里，AUC仪器已经发展到可以容纳不同的检测系统，改进的数据分析已经允许在复杂的混合物中分离离散物种。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，这意味着可以一次扫描多个波长。这是第一次可以在同一个细胞中监测多个物种的沉降，这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将允许我们实时监控多个吸光度曲线，为我们目前无法做到的复杂组装过程提供无与伦比的洞察力。从AUC中获得的信息对大学和制药行业在理解药物靶标相互作用引起的构象变化方面也非常有益（见支持信）。事实上，这是唯一一种能够区分由药物结合引起的构象差异和寡聚化的方法。该应用程序旨在为地方和国家制药部门以及N8大学合作伙伴提供持续的分析超离心，这将是唯一的Optima AUC仪器。目前在英国各所大学只有另外两所，哈维尔大学有一所可供外部使用。新的Optima AUC将为主要用户的项目提供更多令人兴奋的功能，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；确定与多药耐药和肾功能有关的膜蛋白结构；酶的作用机理、生物催化和蛋白质配方的研究。该仪器还将成为BACF培训讲习班的一部分，并将成为下一代生物化学科学家流体动力学培训的一个组成部分。

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.