PDF(Pubmed)
Abstract：
Light-oxygen-voltage (LOV) domains are small photosensory flavoprotein modules that allow the conversion of external stimuli (sunlight) into intracellular signals responsible for various cell behaviors (e.g. phototropism and chloroplast relocation). This ability relies on the light-induced formation of a covalent thioether adduct between a flavin chromophore and a reactive cysteine from the protein environment, which triggers a cascade of structural changes that result in the activation of a serine/threonine (Ser/Thr) kinase. Recent developments in time-resolved crystallography may allow the activation cascade of the LOV domain to be observed in real time, which has been elusive. In this study, we report a robust protocol for the production and stable delivery of microcrystals of the LOV domain of phototropin Phot-1 from Chlamydomonas reinhardtii (CrPhotLOV1) with a high-viscosity injector for time-resolved serial synchrotron crystallography (TR-SSX). The detailed process covers all aspects, from sample optimization to data collection, which may serve as a guide for soluble protein preparation for TR-SSX. In addition, we show that the crystals obtained preserve the photoreactivity using infrared spectroscopy. Furthermore, the results of the TR-SSX experiment provide high-resolution insights into structural alterations of CrPhotLOV1 from Δt = 2.5 ms up to Δt = 95 ms post-photoactivation, including resolving the geometry of the thioether adduct and the C-terminal region implicated in the signal transduction process.
摘要：
光氧电压（LOV）域是小的光感黄素蛋白模块，可将外部刺激（阳光）转化为负责各种细胞行为（例如，向光性和叶绿体重新定位）的细胞内信号。这种能力依赖于黄素发色团和来自蛋白质环境的反应性半胱氨酸之间光诱导的共价硫醚加合物的形成。其触发导致丝氨酸/苏氨酸(Ser/Thr)激酶活化的级联结构变化。时间分辨晶体学的最新发展可能允许实时观察LOV域的激活级联，这是难以捉摸的。在这项研究中,我们报告了一个稳健的方案,用于生产和稳定递送来自莱茵衣藻(CrPhotloV1)的促光生蛋白Phot-1的LOV域微晶,该方案使用用于时间分辨连续同步加速器晶体学(TR-SSX)的高粘度注射器.详细的过程涵盖了所有方面，从样本优化到数据收集，它可以作为TR-SSX的可溶性蛋白制剂的指导。此外,我们表明，使用红外光谱获得的晶体保持光反应性。此外，TR-SSX实验的结果为CrPhotLOV1从Δt=2.5ms到Δt=95ms的光活化后的结构变化提供了高分辨率的见解，包括解析硫醚加合物的几何结构和与信号转导过程有关的C末端区域。
