Dynamic structural changes of the photoactive orange carotenoid protein

本项目旨在研究橙子类胡萝卜素蛋白（OCP）的光活性机制。这种色素-蛋白质复合物是蓝藻中非光化学猝灭（NPQ）的传感器和效应器，并且由于其结构组织，它是研究类胡萝卜素、光活性蛋白和开发新的生物触发系统的方便模型。最近在Scientific Reports、Plant Cell、PNAS、Science和其他顶级期刊上发表的高排名论文证实了这一相关性。尽管近年来取得了成就，但OCP的红色活性形式的结构是未知的，主要是由于无法获得用于X射线结构分析的稳定晶体，因为OCP的结构元素的特征在于高迁移率。在这种情况下，获得结构数据和监测OCP的光化学转化将只能通过使用替代的结构方法：核磁共振（NMR），小角X射线散射（SAXS）和小角中子散射（SANS）在溶液中。为了实现这些目标，我们将使用从蓝细菌集胞藻属PCC 6803产生OCP的大肠杆菌菌株，并为适当的类胡萝卜素辅因子（β-胡萝卜素和叶黄素：海胆酮、玉米黄质、玉米黄质）的掺入提供平台。这些产类胡萝卜素的E.大肠杆菌菌株也是必要的，以获得突变形式的蛋白质，以确定特定氨基酸在光转化过程中的作用，并产生OCP的单个结构域，这对于NMR实验的初始阶段是重要的。用13 C和15 N同位素标记的OCP蛋白的制备将允许我们在NMR光谱中分配来自1H、13 C和15 N的信号，并确定一组光谱参数以计算溶液中的OCP结构。为了研究OCP蛋白质的光循环转化，将使用一套全面的光谱方法：荧光光谱与ps和fs的时间分辨率，相关光谱，红外和拉曼光谱，这将使我们能够将结构数据与蛋白质的光谱特性相关联。SAXS、SANS和交联质谱法（基于化学“交联”绘制相互作用位点，随后对“连接”肽进行质谱鉴定）的组合将产生OCP与荧光恢复蛋白（FRP）蛋白相互作用的基本结构数据，FRP蛋白在NPQ停止期间与OCP相互作用。计划中的研究不仅将提供有关NPQ在蓝藻中的调控机制的基础知识，而且还将提供有关构象变化的动力学知识，以了解决定其独特光谱特征的蛋白质-蛋白质和蛋白质-发色团相互作用的原理。研究结果可用于开发新的蛋白质传感器或光开关系统，用于包括光遗传学在内的生物系统研究。

The project aims to study the mechanisms of photoactivity of orange carotenoid protein (OCP). This pigment-protein complex is a sensor and effector of non-photochemical quenching (NPQ) in cyanobacteria and, because of its structural organization, it is a convenient model for the study of carotenoids, photoactive proteins and for the development of new biological trigger systems. The relevance is confirmed by recent high ranking publications in Scientific Reports, Plant Cell, PNAS, Science and other top-rated journals. In spite of the achievements of recent years, the structure of the red active form of the OCP is unknown, mostly due to inability to obtain stable crystals for X-ray structure analysis, as structural elements of OCP are characterized by high mobility. In this context, obtaining structural data and monitoring of the photochemical transformations of OCP will only be possible by using alternative structural methods: nuclear magnetic resonance (NMR), small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS) in solution. To achieve these goals, we will use Escherichia coli strains that produce OCP from the cyanobacterium Synechocystis sp. PCC 6803 and provide a platform for the incorporation of proper carotenoid cofactors (beta-carotene and xanthophylls: echinenone, canthaxanthin, zeaxanthin). These carotenoid-producing E. coli strains are also necessary to obtain mutant forms of the protein to determine the role of specific amino acids in the process of photoconversion and to produce individual structural domains of OCP, which are important for initial stages of NMR experiments. Preparation of OCP proteins labeled with 13C and 15N isotopes will allow us to assign signals from 1H, 13C and 15N in NMR spectra and to determine a set of spectral parameters to calculate the OCP structure in solution. To study photocyclic transformations of OCP proteins, a comprehensive set of spectroscopic methods will be used: spectrofluorimetry with ps and fs time resolution, correlation spectroscopy, infrared and Raman spectroscopy, which will allow us to correlate structural data with the spectral characteristics of the protein. A combination of SAXS, SANS, and cross-linking mass-spectrometry (mapping interaction sites based on chemical "crosslinking" with subsequent mass spectrometric identification of "linked" peptides) will yield fundamental structural data on the interaction of the OCP with the Fluorescence Recovery Protein (FRP) protein, which interacts with OCP during NPQ cessation. The planned research will not only provide fundamental knowledge about regulatory mechanisms of NPQ in cyanobacteria, but also about the dynamics of conformational changes, in order to understand the principles of protein-protein and protein-chromophore interactions that determine its unique spectral characteristics. The results can be used to develop new protein sensor or photoswitch systems for biological systems research including optogenetics.