Exploring the mycobacterial respiratory supercomplex with Fourier-transformed electrochemistry and cryogenic electron microscopy

利用傅里叶变换电化学和低温电子显微镜探索分枝杆菌呼吸超级复合物

• 批准号: 2885393
• 金额: --
• 依托单位: University of York
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2885393
• 关键词: Exploring; mycobacterial; respiratory; supercomplex; Fourier

Tuberculosis (TB) is a devastating disease caused by the bacteria Mycobacterium tuberculosis and is one of the top 10 causes of death worldwide. Current treatments require a cocktail of antibiotics taken over an extended period with unpleasant side-effects; drug resistance threatens to render even these treatments ineffective. New anti-TB drugs, such as bedaquiline and Q203, target enzymes in the bioenergetic system of TB. This project will focus on delivering a new complementary toolkit to deconvolute the precise chemical mechanism via which these new anti-TB drugs function in order to aid the rational design of new families of antibiotics. The student will combine Fourier-transformed electrochemical methods with structural techniques to probe the kinetic and thermodynamic control of the electron-transfer redox chemistry of the mycobacterial supercomplex. This key enzyme in the bioenergetic system of Mycobacterium tuberculosis takes electrons from menaquinol, using these to convert O2 to water and transducing the released free-energy into the proton-motive force that powers the cell. The mycobacterial supercomplex enzyme is a complex bioinorganic catalyst, it has a set of metal cofactors: heams, Cu centres, and an iron-sulphur cluster, which are used to move electrons in a controlled fashion. It is the unique structure of this enzyme that enables the design of selective drugs that will kill Mycobacterium tuberculosis while not disrupting the equivalent human enzymes. The precise chemistry of the reaction mechanism is not understood and requires in-depth mechanistic study to deconvolute the thermodynamic and kinetic control and map precisely how catalysis is inhibited by drugs such as Q203. The student will use cryo-EM to explore enzyme structure and identify drug binding sites, i.e. precisely where is the antibiotic targeting? What is the orientation of the drug molecule relative to the metal binding sites? Fourier-transformed electrochemistry will be used to study how electrons are moved through the redox-active cofactors that transmit electrons, and to unpick the inhibition mechanism, i.e. how does the drug binding shutdown catalysis? This will be the first time these complementary techniques of cryo-EM, which images biological structures frozen in the vitreous ice state, and Fourier-transformed electrochemistry, which isolates current from cofactor redox chemistry, have been combined to deconvolute antibiotic structure-function chemistry. Objectives-Using an already-prepared strain of Mycobacterium smegmatis (a harmless and fast growing model organism for TB), isolate the enzyme in a catalytically active state.-Use cryo-EM to explore different preparations of the enzyme with the in-house York Glacios electron microscope. -Apply Fourier-transformed electrochemistry to see how electrons move through the enzyme to sustain catalysis.-Combine data from the two approaches to generate quantitative models for how the enzyme functions, using tools from Marcus theory and statistical mechanics.

结核病（TB）是由细菌结核病引起的毁灭性疾病，是全球死亡的十大原因之一。当前的治疗需要长时间服用的抗生素鸡尾酒，并具有不愉快的副作用。耐药性有可能使这些治疗无效。新的抗TB药物，例如Bedaquiline和Q203，靶向结核病生物能系统中的酶。该项目将着重于提供一个新的补充工具包，以解除这些新的抗TB药物作用以帮助新抗生素家族的精确化学机制。该学生将将傅立叶转换的电化学方法与结构技术相结合，以探测分枝杆菌超级复合物的电子转移氧化还原化学的动力学控制和热力学控制。结核分枝杆菌生物能系统中的这种关键酶从梅纳米尔（Menaquinol）吸收了电子，使用这些电子将O2转换为水，并将释放的自由能转化为能为细胞供电的质子动力。分枝杆菌超复合酶是一种复杂的生物有机催化剂，它具有一组金属辅因子：Heam，Cu中心和铁硫簇，用于以受控方式移动电子。这种酶的独特结构使选择性药物的设计将杀死结核分枝杆菌，而不会破坏等效的人类酶。不了解反应机制的精确化学性质，需要深入的机械研究来反应热力学和动力学控制，并准确地绘制了诸如Q203之类的药物抑制催化的方法。学生将使用冷冻EM探索酶结构并识别药物结合位点，即抗生素靶向在哪里？药物分子相对于金属结合位点的方向是什么？傅立叶转换的电化学将用于研究电子如何通过传输电子的氧化还原活性辅助因子移动，并取消抑制机制，即药物结合关闭催化如何？这将是这些冷冻EM的互补技术首次图像在玻璃体冰状态下冷冻的生物结构，而傅立叶转换的电化学已将电流从辅因子氧化还原化学中分离出来，从而将电流结合在一起，从而被合并为反应型抗生素结构 - 抗生素结构 - 抗生素结构 - 抗生素结构。目的是使用已经准备好的分枝杆菌的菌株（一种用于结核病的无害且快速生长的模型生物），将酶隔离为催化活性状态。使用的冷冻EM探索与内部York York Glacios Electron Microscope的酶的不同制剂。 - 采用傅立叶转换的电化学，以了解电子如何通过酶移动以维持催化。从两种方法中结合数据，以使用MARCUS理论和统计力学的工具来生成定量模型，以实现酶的功能。