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) 是由结核分枝杆菌引起的一种毁灭性疾病，是全球十大死因之一。目前的治疗方法需要长期服用多种抗生素，并且会产生令人不快的副作用。耐药性甚至可能导致这些治疗无效。新的抗结核药物，如贝达喹啉和 Q203，以结核生物能量系统中的酶为目标。该项目将重点提供一个新的补充工具包，以解开这些新型抗结核药物发挥作用的精确化学机制，以帮助合理设计新的抗生素家族。学生将把傅里叶变换电化学方法与结构技术相结合，探索分枝杆菌超级复合物电子转移氧化还原化学的动力学和热力学控制。结核分枝杆菌生物能系统中的这种关键酶从甲基萘醌中获取电子，利用这些电子将氧气转化为水，并将释放的自由能转化为为细胞提供动力的质子动力。分枝杆菌超复合酶是一种复杂的生物无机催化剂，它具有一组金属辅助因子：氢原子、铜中心和铁硫簇，用于以受控方式移动电子。正是这种酶的独特结构使得能够设计选择性药物来杀死结核分枝杆菌，同时不破坏等效的人类酶。反应机理的精确化学尚不清楚，需要深入的机理研究来解开热力学和动力学控制，并精确绘制 Q203 等药物如何抑制催化作用。学生将使用冷冻电镜探索酶结构并识别药物结合位点，即抗生素的准确靶向位置在哪里？药物分子相对于金属结合位点的方向是什么？傅里叶变换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，并解开抑制机制，即药物结合关闭催化是如何进行的？这将是首次将冷冻电镜（对玻璃冰状态下冷冻的生物结构进行成像）和傅里叶变换电化学（将电流与辅因子氧化还原化学分离）的互补技术结合起来，以解卷积抗生素结构-功能化学。目标 - 使用已制备的耻垢分枝杆菌菌株（一种无害且快速生长的结核病模式生物），分离处于催化活性状态的酶。 - 使用冷冻电镜与内部 York 一起探索酶的不同制剂格拉西奥斯电子显微镜。 -应用傅里叶变换电化学来了解电子如何穿过酶以维持催化作用。-使用马库斯理论和统计力学的工具，结合两种方法的数据，生成酶如何发挥作用的定量模型。