Structural and Computational Studies of LOV domain proteins and Cryptochromes

描述（由申请人提供）：昼夜节律钟是所有生物王国中真核生物节律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，它根据光线和温度等环境信号进行调节，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。整合转录反馈回路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路中时钟基因的功能，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（苍蝇）的模型系统中对光、氧和电压（LOV）域和隐色素（CRY）这两个对光携带很重要的时钟组件进行结构和计算表征。LOV和CRY成分都是含黄素的光传感器，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结构域是否在不同物种中表现出类似的光化学和光诱导的结构变化，尽管末端残基存在显著差异，我们将通过生化、结构和计算方法研究红杆菌LOV （RLOV）和神经孢子菌Vivid （VVD）中的LOV蛋白在光和暗状态下的变化。目的是确定蛋白质构象变化如何与辅助因子光化学相关，以及这些变化如何诱导信号传播到LOV伴侣和下游时钟组件。RLOV与VVD的相似性表明了蛋白质家族之间的共同机制，并且作为LOV结构域化学的广泛机制具有重要意义。众所周知，果蝇隐色素（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查。dCRY的显著构象变化与光活化有关；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的dCRY结构表征，以阐明其变化。此外，影响时钟控制基因转录的CRY蛋白伴侣——Timeless （TIM）将与dCRY结合进行结构表征，以提供光信号如何转换以激活下游效应的知识。最后，利用x射线晶体学、时间分辨光谱方法的动力学分析和含黄素活性中心的量子化学计算对时钟组件进行结构表征，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。

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.