Structural and Computational Studies of LOV domain proteins and Cryptochromes

LOV 结构域蛋白和隐花色素的结构和计算研究

• 批准号: 8313117
• 负责人: Karen S Conrad
• 金额: $ 4.92万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8313117
• 关键词: ARNT gene; Active Sites; Affect; Aging; Amino Acids; Bacteria; Behavior; Behavioral; Binding; Biochemical; Biological Models; Carbon; Cellular biology; Chemicals; Chemistry; Circadian Rhythms; Complement component C4a; Complex; Computer Simulation; Computing Methodologies; Crystallography; Cues; Cysteine; DNA Sequence Rearrangement; Desire for food; Dimerization; Drosophila genus; Drosophila melanogaster; Electronics; Electrons; Exhibits; Feedback; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Flavins; Genes; Genetic; Genetic Transcription; Goals; Health; Hormones; Human; Induced Mutation; Insecta; Investigation; Kinetics; Knowledge; Laboratories; Lead; Length; Life; Light; Mammals; Mental disorders; Metabolic Pathway; Metabolism; Methods; Modeling; Molecular; Molecular Conformation; N-terminal; Neurospora; Neurospora crassa; Organism; Oxidation-Reduction; Oxygen; Pattern; Periodicity; Photochemistry; Photoreceptors; Physiological; Physiological Processes; Plants; Point Mutation; Production; Property; Protein Family; Proteins; Regulation; Research Proposals; Rhodobacter; Rhodobacter sphaeroides; Signal Transduction; Site; Sleep; Sleep Disorders; Structure; Temperature; Tertiary Protein Structure; Time; Time Perception; Uncertainty; Variant; Work; adduct; chemical reaction; circadian pacemaker; cofactor; computer studies; computerized data processing; cryptochrome; density; design; disorder prevention; electron density; electronic structure; fly; fungus; insight; light entrainment; protein function; quantum; response; sensor; theories; voltage

DESCRIPTION (provided by applicant): Circadian clocks underlie the rhythmic physiological functions and behaviors of eukaryotic organisms in all kingdoms of life. The clock is a widespread cellular mechanism that adjusts to environmental cues like light and temperature and impacts many aspects of our health such as sleep patterns, time-perception, and aging, as well as the treatment and prevention of disease. A complex signaling network that integrates transcriptional feedback loops maintains the rhythm, and although genetics and cell biology studies have identified the functions of clock genes within the loops, the molecular mechanisms of signal processing and propagation are unknown. Two clock components that are important for light entrainment - light, oxygen, and voltage (LOV) domains and Cryptochromes (CRY) - will be structurally and computationally characterized in the model systems of Neurospora (fungi), Rhodobacter (bacteria), and Drosophila (flies) in order to understand how light signals are processes and propagated. The LOV and CRY components are both flavin-containing photosensors that exhibit conformational changes upon light-excitation, but vary in terms of their photochemistry and structure. To determine whether LOV domains exhibit analogous photochemistry and similar light-induced structural changes in different species despite significant differences in terminal residues, Rhodobacter LOV (RLOV) and a LOV protein in Neurospora, Vivid (VVD), will be investigated in both light- and dark-states by biochemical, structural and computational methods. The goal is to determine how protein conformational changes correlate to cofactor photochemistry, and how the changes induce signal propagation to LOV partners and downstream clock components. Similarities of RLOV to VVD would indicate a mechanism shared between protein families, and would be meaningful as a broad mechanism of LOV domain chemistry. Drosophila Cryptochrome (dCRY) is known to transduce light signals associated with circadian clocks. Uncertainties in dCRY photochemistry and signal transduction will also be examined by biochemical, structural, and computational methods. Significant conformational changes of dCRY have been associated with light- activation; thus, structural characterization of dCRY in light-induced and point mutation-designed alternative conformations will be completed using x-ray crystallography to elucidate the changes. Additionally, Timeless (TIM), a protein partner of CRY that impacts transcription of clock controlled genes, will be structurally characterized in combination with dCRY to provide knowledge of how the light signal is converted to activate downstream effects. Ultimately, structural characterization of clock components using x-ray crystallography, kinetic analyses from time-resolved spectroscopic methods, and quantum chemical calculations of the flavin- containing active centers, will provide insight into how flavin photochemistry generates protein conformational changes, and how structural these changes lead to signal propagation. PUBLIC HEALTH RELEVANCE: The circadian clock is a mechanism responsible for coordinating cellular metabolism, the combination of chemical reactions that sustain body function, with a daily cycle. Circadian clocks are present in all kingdoms of life, but in humans the clock impacts a wide variety of physiological processes including sleep patterns, appetite, time perception, hormone production, and aging. This research proposal outlines an investigation of light-sensitive proteins that are critical components of circadian clocks to understand how they work, their structural components and features, and how they communicate with other proteins.

描述（由申请人提供）：生物钟是所有生命领域真核生物节律性生理功能和行为的基础。生物钟是一种广泛存在的细胞机制，它可以适应光和温度等环境因素，并影响我们健康的许多方面，例如睡眠模式、时间感知和衰老，以及疾病的治疗和预防。整合转录反馈环的复杂信号网络维持着节律，尽管遗传学和细胞生物学研究已经确定了环内时钟基因的功能，但信号处理和传播的分子机制尚不清楚。对光夹带很重要的两个时钟成分——光、氧和电压（LOV）域和隐花色素（CRY）——将在脉孢菌（真菌）、红细菌（细菌）和果蝇（苍蝇）的模型系统中进行结构和计算表征，以便了解光信号是如何处理和传播的。 LOV 和 CRY 组件都是含黄素的光传感器，在光激发下表现出构象变化，但其光化学和结构有所不同。为了确定不同物种的 LOV 结构域是否表现出类似的光化学和类似的光诱导结构变化，尽管末端残基存在显着差异，将通过生化、结构和计算方法在光状态和暗状态下研究红细菌 LOV (RLOV) 和脉孢菌中的 LOV 蛋白，Vivid (VVD)。目标是确定蛋白质构象变化如何与辅因子光化学相关，以及这些变化如何诱导信号传播到 LOV 伙伴和下游时钟组件。 RLOV 与 VVD 的相似性表明蛋白质家族之间共享的机制，并且作为 LOV 结构域化学的广泛机制是有意义的。果蝇隐花色素 (dCRY) 已知可以传导与生物钟相关的光信号。 dCRY 光化学和信号转导的不确定性也将通过生物化学、结构和计算方法进行检查。 dCRY 的显着构象变化与光激活有关；因此，将使用 X 射线晶体学来完成光诱导和点突变设计的替代构象中 dCRY 的结构表征，以阐明这些变化。此外，Timeless (TIM) 是 CRY 的一种蛋白质伴侣，可影响时钟控制基因的转录，它将与 dCRY 结合进行结构表征，以提供有关光信号如何转换以激活下游效应的知识。最终，使用 X 射线晶体学对时钟组件进行结构表征、时间分辨光谱方法的动力学分析以及含黄素活性中心的量子化学计算，将深入了解黄素光化学如何产生蛋白质构象变化，以及这些结构变化如何导致信号传播。 公共健康相关性：生物钟是一种负责协调细胞代谢的机制，细胞代谢是维持身体功能的化学反应与日常周期的结合。生物钟存在于生命的各个领域，但在人类中，生物钟影响多种生理过程，包括睡眠模式、食欲、时间感知、激素产生和衰老。该研究计划概述了对光敏蛋白的研究，光敏蛋白是生物钟的关键组成部分，以了解它们的工作原理、结构成分和特征，以及它们如何与其他蛋白沟通。