Regulation of Substrate Binding in the bc1 Complex

bc1 复合物中底物结合的调节

• 批准号: 10203271
• 负责人: Oleksandr Kokhan
• 金额: $ 40.21万
• 依托单位: JAMES MADISON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10203271
• 关键词: Affinity; Amines; Antimalarials; Apoptosis; Binding; Binding Sites; Calorimetry; Cardiolipins; Cardiovascular Diseases; Cations; Charge; Chemicals; Complex; Crystallization; Cytochrome bc1 Complex; Cytochrome c1; Cytochromes b; Data; Detergents; Diabetes Mellitus; Disease; Down-Regulation; Electron Transport Complex III; Environment; Enzymes; Future Generations; Genes; Genome; Hydrogen Bonding; In Vitro; Institution; Ionic Strengths; Kinetics; Lasers; Link; Lipids; Liver; Location; Lung; Lys-Asp; Lysine; Malignant Neoplasms; Measures; Membrane; Membrane Lipids; Messenger RNA; Mitochondria; Mitochondrial Myopathies; Modification; Molecular; Mutation; Neurodegenerative Disorders; Optics; Ovarian; Oxidation-Reduction; Oxidoreductase; Premature aging syndrome; Production; Proteins; Protons; Quinones; Reactive Oxygen Species; Recording of previous events; Regulation; Research Training; Resolution; Respiration; Rieske iron-sulfur protein; Roentgen Rays; Role; Spectrum Analysis; Structure; Students; Techniques; Testing; Thermodynamics; Time; Up-Regulation; Variant; Work; Yeasts; atovaquone; base; biomedical scientist; carboxylate; career; crosslink; cytochrome c; dimer; early onset; enzyme structure; enzyme substrate complex; interdisciplinary approach; interest; kidney cell; malignant breast neoplasm; molecular dynamics; monomer; nanodisk; nervous system disorder; optic nerve disorder; programs; response; stoichiometry; ubiquinol; undergraduate research; undergraduate student

Abstract Ubiquinol-cytochrome c oxidoreductase (bc1 complex, complex III) is a key membrane enzyme involved in respiration. It is known to be one of the major producers of reactive oxygen species (ROS) in mitochondria. Our overarching hypothesis is that there are at least three overlooked bc1 regulations mechanisms involving cytochrome (cyt) c1. Specifically, our Aim I is to use a combination of computational and experimental techniques to test anticooperative substrate binding in the bc1 complex. This effect was suggested based on available X-ray crystal structures but was not experimentally tested. Our preliminary molecular dynamics (MD) simulations provide us with a testable structural mechanism which we will test experimentally. Our Aim II is to establish the role of naturally occurring trimethylation of Lys-77 by a unique and specific cyt c lysine methylatransferase (Ctm1) in yeast. Our hypothesis that this posttranslation modification regulates the strength of cation-pi interaction between Lys-77 of cyt c and universally conserved in species with Ctm1p Phe-132 in cyt c1. Finally, our Aim III is focused on testing a hypothesis that lipid membrane composition and lipid charge can regulate substrate binding affinity in the bc1 complex. This project will use a multi-pronged approached combining computational and experimental techniques to predict molecular level bc1 regulation mechanisms and to test them experimentally. We will use long all-atom MD simulations of bc1 in different lipid environments to predict structural changes associated with different occupancy of the substrate binding sites and to guide our experimental work on detergent-solubilized and nanodisc-embedded bc1. We will use isothermal calorimetry (ITC) to test substrate binding in vitro, and to measure binding stoichiometries, association constants, and thermodynamic parameters as a function of ionic strength and lipid charge. We will use small-angle X-ray scattering (SAXS) to independently verify the ITC results, to confirm the locations of substrate binding sites, and to construct low-resolution solution-state structures of the enzyme-substrate complexes. Laser-induced time-resolved optical spectroscopy will be used to measure changes in the charge transfer rates as response to changes in lipid environment and substrate binding regulation. Finally, we will use kinetic spectroscopy to study the roles of lipid membranes and intermonomer interactions within the bc1 complex dimer on the catalytic turnover rates and the rate of ROS production. Overall, this interdisciplinary approach will advance understanding of cyt bc1 regulation and will test the three predicted regulation mechanisms. In addition, this project will directly support each year research training of 4 undergraduate students interested in pursuing biomedical careers.

抽象的 泛醇-细胞色素 C 氧化还原酶（bc1 复合物，复合物 III）是一种关键的膜酶 参与呼吸。众所周知，它是活性氧的主要生产者之一 （ROS）在线粒体中。我们的总体假设是至少存在三个 忽视了涉及细胞色素 (cyt) c1 的 bc1 调控机制。具体来说，我们的 目标 I 是结合计算和实验技术来测试 bc1 复合物中的抗合作底物结合。该效果是基于以下建议的 可用的 X 射线晶体结构，但未经实验测试。我们的初步分子 动力学（MD）模拟为我们提供了一个可测试的结构机制，我们将对其进行测试 实验性地。我们的目标 II 是确定 Lys-77 自然发生的三甲基化的作用 由酵母中独特且特异性的细胞色素 c 赖氨酸甲基转移酶 (Ctm1) 产生。我们的假设是这 翻译后修饰调节细胞色素 Lys-77 之间阳离子-π 相互作用的强度 c 并且在 cyt c1 中具有 Ctm1p Phe-132 的物种中普遍保守。最后，我们的目标三是 重点测试脂质膜组成和脂质电荷可以调节的假设 bc1 复合物中的底物结合亲和力。该项目将采用多管齐下的方式 结合计算和实验技术来预测分子水平 bc1 调控 机制并通过实验对其进行测试。我们将使用 bc1 的长全原子 MD 模拟 不同的脂质环境来预测与不同占据相关的结构变化 底物结合位点并指导我们关于洗涤剂溶解和 纳米圆盘嵌入式 bc1。我们将使用等温量热法（ITC）来测试底物结合 体外，并测量结合化学计量、缔合常数和热力学 参数作为离子强度和脂质电荷的函数。我们将使用小角度 X 射线 散射（SAXS）独立验证ITC结果，确认基材的位置 结合位点，并构建酶底物的低分辨率溶液态结构 复合物。激光诱导时间分辨光谱将用于测量变化 电荷转移速率响应脂质环境和底物结合的变化 规定。最后，我们将使用动力学光谱来研究脂质膜和 bc1复合物二聚体内单体间相互作用对催化周转率和 ROS 产生率。总的来说，这种跨学科方法将增进对细胞色素的理解 bc1 调节并将测试三种预测的调节机制。另外，这个项目 每年将直接支持4名感兴趣的本科生的研究培训 追求生物医学事业。