Structural and Computational Studies of LOV domain proteins and Cryptochromes

描述(申请人提供)：生物钟是真核生物在所有生命王国中有节奏的生理功能和行为的基础。时钟是一种广泛存在的细胞机制，它可以根据光和温度等环境信号进行调整，并影响我们健康的许多方面，如睡眠模式、时间感知、衰老以及疾病的治疗和预防。一个整合转录反馈环的复杂信号网络维持着节奏，尽管遗传学和细胞生物学研究已经确定了时钟基因在环中的功能，但信号处理和传播的分子机制尚不清楚。为了了解光信号是如何处理和传播的，我们将在神经孢子菌(真菌)、红杆菌(细菌)和果蝇(果蝇)的模型系统中从结构和计算上表征对光携带至关重要的两个时钟成分-光、氧和电压(LOV)结构域和隐色素(CRY)。LOV和CRY组分都是含有黄素的光传感器，在光激发下表现出构象变化，但在光化学和结构方面有所不同。为了确定LOV结构域在不同物种中是否表现出相似的光化学和相似的光诱导结构变化，尽管末端残基存在显著差异，我们将用生化、结构和计算方法研究红细菌LOV(RLOV)和神经孢子菌中的LOV蛋白Vivid(VVD)在光和暗状态下的变化。我们的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存在共同的机制，作为LOV结构域化学的一种广泛机制具有重要意义。众所周知，果蝇隐花色素(DCRY)可以传递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化、结构和计算方法进行检验。DCRY的显著构象变化与光激活有关，因此，光诱导和点突变设计的替代构象中dCRY的结构表征将用X射线结晶学来解释这些变化。此外，影响时钟控制基因转录的CRY的蛋白质伙伴Timless(TIM)将与dCRY结合在一起进行结构表征，以提供光信号如何转换以激活下游效应的知识。最终，使用X射线结晶学对时钟组件的结构表征、来自时间分辨光谱方法的动力学分析以及对含有黄素的活性中心的量子化学计算，将提供对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的洞察。 与公共健康相关：生物钟是一种负责协调细胞新陈代谢的机制，细胞新陈代谢是维持身体功能的化学反应与日常周期的组合。生物钟存在于所有生命王国中，但在人类中，生物钟影响着各种各样的生理过程，包括睡眠模式、食欲、时间感知、荷尔蒙分泌和衰老。这项研究计划概述了对光敏蛋白质的研究，这些蛋白质是生物钟的关键组成部分，以了解它们是如何工作的，它们的结构成分和特征，以及它们如何与其他蛋白质通信。

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.