STRUCTURAL CHARACTERIZATION OF NATURAL AND DESIGNED PHOTOSENSORS OF THE LIGHT

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 对环境刺激的感知和整合对所有生物来说都是基本的，并且经常由多才多艺的Per-Arnt-Sim(Pas)家族成员调节，该家族存在于所有生命王国中。PAS家族的一个亚类是光-氧-电压(LOV)蛋白，它作为辅因子与黄素单核苷酸(FMN)结合。在蓝光照射下，LOV光电传感器在半胱氨酸残基和FMN之间形成共价键。我们的研究旨在了解和控制LOV蛋白中信号的产生和传播。研究的主线有两条。首先，我们将研究天然LOV蛋白中的光信号。我们的研究集中在光如何影响蛋白质的结构和动力学，以及初始信号是如何传递到下游效应器的。LOV结构域及其共轭效应器的高分辨率结构将提供对这些过程的机械洞察。我们还计划通过冷冻捕获技术或时间分辨X射线结晶学来获得关于这些蛋白质的光激活状态的结构信息。后者还允许在光循环期间访问瞬时填充的中间体。在第二种方法中，将利用分子生物学和体外进化技术设计基于LOV结构域的人造蛋白质。通过共价连接效应蛋白和LOV结构域，我们希望将它们的功能置于光控制下。稳定和瞬时填充状态的结构信息对于阐明这些蛋白质的分子机制是必不可少的。新型光调控蛋白的成功设计将提高我们对信号传递过程的理解，并可用于生物技术和其他应用。与在天然蛋白质上获得的结果进行比较，突出了相似和不同，这应该会提高我们对LOV结构域中信号传递的了解。我们的研究也可能被证明与更大的PAS家族的信号转导相关。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Perception and integration of environmental stimuli are elemental for all living organisms and are frequently mediated by members of the versatile Per-Arnt-Sim (PAS) family which occur in all kingdoms of life. A subclass of the PAS family are the light-oxygen-voltage (LOV) proteins which bind flavin mononucleotide (FMN) as a cofactor. Upon blue light illumination LOV photosensors form a covalent bond between a cysteine residue and FMN. Our research aims at understanding and controlling signal generation and propagation in LOV proteins. Two main lines of research are pursued. First, we will study light signaling in natural LOV proteins. Our research focuses on how protein structure and dynamics are influenced by light and how the initial signal is relayed to downstream effectors. High-resolution structures of LOV domains and their conjugate effectors will provide mechanistic insight into these processes. We also plan to obtain structural information on the photoactivated state of these proteins either by freeze trapping techniques or time-resolved X-ray crystallography. The latter also allows to access transiently populated intermediates during the photocycle. In a second approach artificial proteins based on LOV domains will be designed using molecular biology and in vitro evolution techniques. By covalently linking effector proteins to LOV domains, we want to place their function under light control. Structural information both on stable and transiently populated states will be essential for elucidating the molecular mechanism of these proteins. The successful design of novel light-regulated proteins would improve our understanding of the signaling process and could be used in biotechnological and other applications. Comparison to results obtained on natural proteins highlights similarities and differences which should improve our knowledge about signaling in LOV domains. Our research could also prove relevant for signal transduction in the larger PAS family.