Administrative Supplement to Purchase a High-Performance Computing System

摘要 泛喹酚-细胞色素c氧化还原酶(bc1复合体，复合体III)是细胞膜的关键酶。 与呼吸有关的。众所周知，它是产生活性氧的主要物种之一。 (ROS)。我们最重要的假设是至少有三个 忽视了涉及细胞色素(Cyt)c1的Bc1调节机制。具体地说，我们的 目标1是使用计算和实验技术的组合来测试 Bc1复合体中的反合作底物结合。这种效应是基于以下因素提出的 可用的X射线晶体结构，但没有经过实验测试。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 试验性的。我们的目标二是确定自然发生的Lys-77三甲基化的作用 通过酵母中一种独特和特异的细胞色素C-赖氨酸甲基转移酶(Ctm1)。我们的假设是这 翻译后修饰调节Cyt细胞Lys-77之间阳离子-pi相互作用的强度 C，在Cyt C1中与CTM 1p Phe-132同源，在物种中普遍保守。最后，我们的目标三是 重点检验了脂膜成分和脂电荷可以调节的假说 BC1复合体中的底物结合亲和力。这个项目将采用多管齐下的方法。 结合计算和实验技术预测分子水平的bc1调控 并对其进行实验测试。我们将使用长的全原子MD模拟Bc1在 不同的脂质环境来预测与不同的居住环境相关的结构变化 底物结合部位，并指导我们的实验工作，洗涤剂溶解和 纳米盘-嵌入Bc1。我们将使用等温量热法(ITC)来测试衬底结合 体外，并测量结合化学计量、结合常数和热力学 参数作为离子强度和脂类电荷的函数。我们将使用小角X射线 散射(SAXS)以独立验证ITC结果，以确定衬底的位置 结合位置，并构建酶-底物的低分辨率溶液状态结构 复合体。激光诱导的时间分辨光谱学将被用来测量变化 电荷转移率对脂质环境和底物结合变化的响应 监管。最后，我们将使用动力学光谱来研究脂膜和 Bc1络合二聚体中单体间相互作用对催化转化率和反应速率的影响 ROS产生率。总体而言，这种跨学科的方法将促进对细胞免疫技术的理解。 Bc1调控，并将检验预测的三种调控机制。此外，这个项目 将直接支持每年4名有兴趣的本科生进行科研培训 追求生物医学事业。

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 1 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 Ctm 1p 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 approach 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.