Structures, Dynamics and Signaling Mechanisms of Bacteriophytochromes

描述(申请人提供)：光是生命有机体的基本环境信号。光感受器将光信号转化为生化和生物信号，最终调节一系列重要的生理过程，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了解光感受器的信号机制。我们结合结晶学、X射线溶液散射、光谱和生化方法研究了红光/远红光感受器细菌藻色素(BphP)的结构和信号机制。双酚P吸收并响应可见太阳光谱中650 nm至900 nm之间的长波长范围内的光子。典型的BphP在红光吸收(PR)和远红光吸收(PFR)状态之间进行可逆的光转换，其中其C末端组氨酸激酶(HK)结构域经历光依赖的自动磷酸化，然后通过双组分信号通路将磷酸基团传递给下游的反应调节因子。本研究的核心是关于BPP中的信令的三个核心问题。1)当吸收一个光子时，发色团的构象发生了什么变化？2)光诱导蛋白质部分的结构信号的性质是什么？3)局部结构信号是如何从发色团结合部位传递到空间距离较远的HK的活性部位的？我们通过在广泛的时间和长度尺度上研究三个具有代表性的BphP中光诱导的分子事件的结构、动力学和动力学路径来解决这些问题。我们应用静态结晶学和诱变来确定关键结构元素和不同信号状态下的相互作用。我们进行了泵浦探测实验，通过晶体和光活性双酚P溶液的X射线散射来引发和跟踪光反应，以直接观察室温下光诱导的结构变化。因此，我们在分子水平上探索了红光信号转化为生物信号的机制。此外，BphP的长程信号转导原理将对理解更广泛的、模块化的信号蛋白(如化学受体)的分子机制具有更广泛的意义。由于BphPs的作用光谱范围与人类的治疗光学窗口一致，因此BphPs作为红色荧光蛋白在深部组织荧光成像中具有巨大的潜力，并作为遗传编码工具用于体内功能的光学操作。我们的发现将作为一个结构框架，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.