Enzyme mechanisms studied by single molecule optical sensing

该项目将结合尖端的物理和生物化学方法。泛素化反应将使用等离子体增强的回音廊模式(WGM)微腔传感来研究。这是第一个能够直接监测蛋白质等单个生物分子内部结构变化的光学技术。一个主要目标将是确定每种类型的泛素连接是否具有唯一的WGM签名。Bath的Bagby和Whitley博士进行的生化和化学生物学实验将包括泛素化分析，以及泛素和酶(连接酶和脱泛素酶)的产生，这些酶经过修饰后可固定在用于WGM传感的金纳米颗粒上。Exeter将与Vollmer教授的团队一起进行泛素链组装的单分子WGM传感研究。泛素化是一种重要的翻译后修饰，在大多数(如果不是全部)真核细胞中，泛素化参与了蛋白质功能的许多调节和调节过程；泛素化在许多疾病中都出现了错误。泛素化涉及一种称为泛素的小蛋白的一个分子或多个分子与目标蛋白(底物)的共价连接。如下所述，可以组装不同类型的泛素链；这一点很重要，因为链类型决定了泛素化的生物学结果(例如底物蛋白质是否被降解，或者其功能或位置是否受到影响)，但目前很难确定链类型。本研究的目的是建立一种快速、准确、易用的方法来鉴定任何E3泛素连接酶组装的泛素链的类型，同时为泛素化的机制和动力学提供新的见解。泛素连接酶催化的泛素化是泛素通过泛素的C端羧基与底物的N端胺或赖氨酸残基的氨基之间形成异肽键而共价连接到蛋白质底物上。在许多情况下，泛素分子链组装在底物蛋白质上，泛素单体通过七个赖氨酸或N-末端中的任何一个连接到链上。泛素链可以包括单一类型的赖氨酸键或混合键，每个键产生不同程度的灵活性和构象状态。由于链类型决定生物学结果，该项目将对E3连接酶功能和所产生的表型之间的关系提供详细的新见解，因此可能会促进对特定泛素连接酶与健康和疾病之间关系的理解。

The project will involve a combination of cutting edge physical and biochemical methods. Ubiquitination reactions will be studied using plasmonically enhanced whispering gallery mode (WGM) microcavity sensing. This is the first optical technique capable of directly monitoring structural changes within individual biomolecules such as proteins. A major aim will be establishing whether each type of ubiquitin linkage has a unique WGM signature. Biochemical and chemical biology experiments with Drs Bagby and Whitley at Bath will include ubiquitination assays, and production of ubiquitin and enzymes (ligases and deubiquitinases) modified for immobilisation on gold nanoparticles that are used in WGM sensing. Single molecule WGM sensing studies of ubiquitin chain assembly will be conducted at Exeter with Professor Vollmer's group. Ubiquitination is an important post-translational modification involved in modulation and regulation of protein function in many processes in most, if not all, eukaryotic cell types; ubiquitination goes awry in numerous diseases. Ubiquitination involves the covalent attachment to a target protein (the "substrate") of one molecule, or multiple molecules in chains, of a small protein called ubiquitin. As described below, different types of ubiquitin chains can be assembled; this is important because chain type determines the biological outcome of ubiquitination (e.g. whether the substrate protein is degraded, or its function or location is affected), but currently it is difficult to determine chain type. The aim is to develop a rapid, accurate and user-friendly method to identify the type of ubiquitin chain assembled by any E3 ubiquitin ligase, at the same time providing new insights into ubiquitination mechanism and kinetics.Ubiquitination, catalysed by ubiquitin ligases, involves covalent conjugation of ubiquitin to the protein substrate via formation of an isopeptide bond between ubiquitin's C-terminal carboxylate and the substrate's N-terminal amine or -amino group of a lysine residue. In many cases a chain of ubiquitin molecules is assembled on a substrate protein whereby a ubiquitin monomer is linked to the chain via any one of seven lysines or N-terminus. Ubiquitin chains can involve a single type of lysine linkage or mixed linkages, with each linkage producing a different degree of flexibility and repertoire of conformational states. Since chain type determines biological outcome, this project will provide detailed new insights into the relationships between E3 ligase function and resulting phenotype, and will therefore potentially advance understanding of the relationships between particular ubiquitin ligases and health and disease.