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

结核病（TB）是由结核分枝杆菌（Mycobacterium tuberculosis）引起的一种毁灭性疾病，是全球十大死亡原因之一。目前的治疗需要长时间服用抗生素的鸡尾酒，副作用令人不快;耐药性甚至有可能使这些治疗无效。新的抗结核药物，如贝达喹啉和Q203，靶向结核病生物能量系统中的酶。该项目的重点是提供一个新的补充工具包，以解卷积这些新的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。学生将结合联合收割机傅立叶变换的电化学方法与结构技术，探讨分枝杆菌超复合物的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量系统中的这种关键酶从甲基喹啉中获取电子，利用这些电子将O2转化为水，并将释放的自由能转换为质子动力，为细胞提供动力。分枝杆菌超复合酶是一种复杂的生物无机催化剂，它有一组金属辅因子：heams，Cu中心和铁硫簇，用于以受控的方式移动电子。正是这种酶的独特结构使得能够设计出选择性药物，这些药物将杀死结核分枝杆菌，而不会破坏等同的人类酶。反应机理的精确化学尚不清楚，需要深入的机理研究来解卷积热力学和动力学控制，并精确绘制催化如何被药物如Q203抑制。学生将使用cryo-EM来探索酶的结构和确定药物结合位点，即抗生素的靶向位置。药物分子相对于金属结合位点的方向是什么？傅立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因子移动，并解开抑制机制，即药物结合如何关闭催化？这将是第一次将这些cryo-EM的互补技术（它对冻结在玻璃冰状态下的生物结构进行成像）和傅立叶变换电化学（它将电流与辅因子氧化还原化学分离）结合起来，以解卷积抗生素结构-功能化学。目的-使用已经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cryo-EM，通过内部约克Glacios电子显微镜探索酶的不同制备。- 应用傅立叶变换电化学来观察电子如何通过酶来维持催化。使用Marcus理论和统计力学的工具，将来自两种方法的联合收割机数据结合起来，生成酶功能的定量模型。

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.