Structural and Computational Studies of LOV domain proteins and Cryptochromes

描述（由申请人提供）：生物钟是所有生命王国中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，可以根据光线和温度等环境线索进行调整，并影响我们健康的许多方面，如睡眠模式，时间感知和衰老，以及疾病的治疗和预防。一个复杂的信号网络整合了转录反馈环，维持着这种节奏，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。两个时钟组件是重要的光夹带-光，氧，电压（LOV）域和隐花色素（CRY）-将结构和计算的特点，在模型系统的脉孢菌（真菌），红细菌（细菌），果蝇（苍蝇），以了解光信号是如何处理和传播。LOV和CRY组分都是含有黄素的光传感器，其在光激发时表现出构象变化，但在它们的光化学和结构方面有所不同。为了确定LOV结构域是否表现出类似的光化学和类似的光诱导的结构变化，在不同的物种，尽管在末端残基，红细菌LOV（RLOV）和一个LOV蛋白链孢霉，生动（VVD）的显着差异，将在光和暗状态下的生物化学，结构和计算方法进行了研究。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）是已知的抑制与生物钟相关的光信号。在dCRY光化学和信号转导中的不稳定性也将通过生物化学，结构和计算方法进行检查。dCRY的显著构象变化与光活化相关;因此，将使用X射线晶体学完成光诱导和点突变设计的替代构象中dCRY的结构表征，以阐明变化。此外，Timeless（TIM）是CRY的一种蛋白质伴侣，影响时钟控制基因的转录，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。最终，使用X射线晶体学的时钟组件的结构表征，从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，将提供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.