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Enzyme mechanisms studied by single molecule optical sensing

单分子光学传感研究酶机制

基本信息

批准号：
2401633
负责人：
金额：
--
依托单位：
UNIVERSITY OF EXETER
依托单位国家：
英国
项目类别：
Studentship
财政年份：
2020
资助国家：
英国
起止时间：
2020 至无数据
项目状态：
未结题

来源：
https://gtr.ukri.org/projects?ref=studentship-2401633
关键词：
Enzyme mechanisms studied single molecule

项目摘要

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.

该项目将涉及尖端物理和生化方法的结合。泛素化反应将研究使用等离子体增强低语廊模式（WGM）微腔传感。这是第一个能够直接监测单个生物分子（如蛋白质）结构变化的光学技术。一个主要的目标将是确定是否每一种类型的泛素连锁具有独特的WGM特征。巴格比博士和惠特利博士在巴斯大学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（连接酶和去泛素酶）的生产，这些酶被修饰为固定在用于WGM传感的金纳米颗粒上。泛素链组装的单分子WGM传感研究将在埃克塞特大学与Vollmer教授的小组一起进行。泛素化是一种重要的翻译后修饰，在大多数（如果不是全部）真核细胞类型的许多过程中参与蛋白质功能的调节和调节；泛素化在许多疾病中出错。泛素化涉及到一种叫做泛素的小蛋白质的一个分子或多个分子链与目标蛋白质（“底物”）的共价附着。如下所述，可以组装不同类型的泛素链；这一点很重要，因为链的类型决定了泛素化的生物学结果（如底物蛋白是否被降解，或其功能或位置是否受到影响），但目前很难确定链的类型。目的是建立一种快速、准确和用户友好的方法来鉴定任何E3泛素连接酶组装的泛素链的类型，同时为泛素化机制和动力学提供新的见解。泛素化由泛素连接酶催化，通过在泛素的c端羧酸和底物的n端胺或赖氨酸残基之间形成异肽键，将泛素与蛋白质底物共价结合。在许多情况下，泛素分子链被组装在底物蛋白上，泛素单体通过7个赖氨酸或n端中的任何一个连接到该链上。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库。由于链型决定生物学结果，该项目将为E3连接酶功能与结果表型之间的关系提供详细的新见解，因此将潜在地促进对特定泛素连接酶与健康和疾病之间关系的理解。