Structures, Dynamics and Signaling Mechanisms of Bacteriophytochromes

描述（申请人提供）：光是生物体的基本环境信号。光感受器将光信号转化为生化和生物信号，最终调节广泛的重要生理过程，如光合作用、昼夜节律和视觉。我们的长期目标是在分子水平上理解光感受器的信号机制。我们结合晶体学、x射线溶液散射、光谱学和生化方法来研究红光/远红光光感受器细菌色素（BphPs）的结构和信号传导机制。BphPs吸收并响应可见光光谱650 - 900纳米波长范围内的光子。典型的BphP在红光吸收（Pr）和远红光吸收（Pfr）状态之间可逆地进行光转化，其中其c端组氨酸激酶（HK）结构域经历光依赖性自磷酸化，然后在双组分信号通路中将磷酸基团传递给下游响应调节因子。本研究的核心是关于BphPs信号传导的三个核心问题。1)在吸收光子时，在发色团中触发了什么构象变化？2)光诱导蛋白片段结构信号的本质是什么？3)局部结构信号如何从发色团结合位点传递到空间遥远的HK的活性位点？我们通过在大范围的时间和长度尺度上研究三个代表性BphPs的光诱导分子事件的结构、动力学和动力学途径来解决这些问题。我们应用静态晶体学和诱变来识别不同信号状态下的关键结构元件和相互作用。我们利用光活性BphPs晶体和溶液的x射线散射，进行了泵浦探针实验，以引发和跟踪光反应，直接观察室温下光诱导的结构变化。因此，我们探索了红光信号在分子水平上转化为生物信号的机制。此外，BphPs的远程信号转导原理将对理解更广泛的模块化信号蛋白（如化学受体）的分子机制具有更广泛的意义。由于BphPs的作用谱范围与人类的治疗光学窗口一致，因此BphPs作为深层组织荧光成像的红色荧光蛋白和作为体内功能光学操作的遗传编码工具具有很大的潜力。我们的发现将作为一个结构框架来指导基于bphp的生物医学应用的进一步发展。

DESCRIPTION (provided by applicant): Light is a fundamental environmental signal for living organisms. Photoreceptors convert light signals into biochemical and biological signals that ultimately regulate a wide range of important physiological processes such as photosynthesis, circadian rhythm and vision. Our long-term goal is to understand the signaling mechanisms of photoreceptors at the molecular level. We integrate crystallography, X-ray solution scattering, spectroscopic and biochemical approaches to investigate structures and signaling mechanisms of the red-/far-red-light photoreceptors, bacteriophytochromes (BphPs). BphPs absorb and respond to photons in the long wavelength range of the visible solar spectrum between 650nm and 900nm. A typical BphP photo-converts reversibly between red-light- absorbing (Pr) and far red-light-absorbing (Pfr) states, in which its C-terminal histidine kinase (HK) domain undergoes light-dependent auto-phosphorylation and then relays the phosphate group to a downstream response regulator in a two-component signaling pathway. At the center of this research are three core questions on signaling in BphPs. 1) What conformational changes are triggered in the chromophore upon absorbing a photon? 2) What is the nature of light-induced structural signals in the protein moiety? 3) How are local structural signals transmitted from the chromophore-binding site to the active site of the spatially distant HK? We address these questions by examining structures, dynamics and kinetic pathways of light-induced molecular events in three representative BphPs, on a wide range of time and length scales. We apply both static crystallography and mutagenesis to identify key structural elements and interactions in distinct signaling states. We conduct pump-probe experiments to initiate and follow photoreactions by X-ray scattering from both crystals and solutions of photoactive BphPs, to directly observe light-induced structural changes at room temperature. We thus explore the mechanism by which a red light signal is converted into a biological signal at the molecular level. In addition, the principles of long-range signal transduction in BphPs will have broader implications for understanding molecular mechanisms of more widespread, modular signaling proteins such as chemoreceptors. Since the range of the action spectrum of BphPs coincides with the therapeutic optical window for humans, BphPs have great potential as red fluorescent proteins for deep tissue fluorescent imaging and as genetically encoded tools for optical manipulation of in vivo functions. Our findings will serve as a structural framework to guide further development of BphP-based biomedical applications.