Structures, Dynamics and Signaling Mechanisms of Modular Photoreceptors

模块化感光器的结构、动力学和信号机制

• 批准号: 10219257
• 负责人: XIAOJING YANG
• 金额: $ 37.83万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10219257
• 关键词: Active Sites; Address; Adopted; Affect; Allosteric Regulation; Architecture; Arithmetic; Award; Basic Science; Bilin; Binding; Biochemical; Biochemistry; Biological; Biological Assay; Biological Models; Biomedical Research; Biotechnology; Cell membrane; Chemicals; Chemoreceptors; Chimera organism; Color Perception; Complex; Coupled; Coupling; Cryoelectron Microscopy; Crystallization; Crystallography; Data Set; Distant; Electron Microscopy; Elements; Engineering; Event; Family; Foundations; Generations; Goals; Image; Individual; Integral Membrane Protein; Joints; Length; Ligand Binding; Ligands; Light; Lighting; Logic; Mechanoreceptors; Methods; Methylation; Molecular; Molecular Conformation; Motion; Mutagenesis; Myelin P2 Protein; Nutrient; Organism; Output; Oxygen; Perception; Phosphorylation; Phosphotransferases; Photoreceptors; Phytochrome; Play; Process; Proteins; Research; Resolution; Role; Sensory; Signal Transduction; Signaling Protein; Site; Site-Directed Mutagenesis; Slide; Spectrum Analysis; Structure; System; Temperature; Therapeutic; Torque; Vertebral column; Work; X ray diffraction analysis; X-Ray Crystallography; base; biophysical properties; biophysical techniques; chromophore; cryogenics; dimer; experimental study; extracellular; operation; particle; pi bond; protein-histidine kinase; reconstruction; response; scaffold; sensor

Project Summary The ability to sense and respond to complex environmental signals such as light, oxygen and nutrients is critical for survival and adaptation of living organisms. Many signaling proteins adopt multi-domain modular architecture to accomplish the perception of input signals and the generation of an output biological response within the same protein molecule. One example of widespread modular systems is offered by bilin-based photoreceptors in the phytochrome superfamily. Since light can readily penetrate the cell membrane, these soluble multi-domain photoreceptors offer excellent model systems for studying the still elusive mechanism of long-range signaling and allosteric regulation in modular signaling proteins such as chemoreceptors and mechanoreceptors. Our long-term goal is to understand how modular photoreceptors perceive, integrate, and transduce signals at the molecular level. To attack this goal, we adopt an integrated approach of biochemistry, spectroscopy, crystallography and cryoEM single particle reconstruction, with a main thrust on dynamic crystallography, which enables direct observation of structural responses at atomic resolution. In this proposal, we use two dual-sensor photoreceptors to represent two major types of bilin-binding photoreceptors. Both are sensory histidine kinases that feature different color perception and distinct signaling logic in response to light or chemical signals. Previously, we have obtained abundant structural information on various isolated domains by both static and dynamic crystallography. In this proposal, we will investigate the molecular mechanisms of signal integration and allosteric activation in full-length proteins where the sensor and effector domains are coupled. Specifically, we will capture structural changes in each sensory site by introducing perturbations via ligand soaking and light illumination. We will examine how the structural signals are initiated and how they propagate through the protein framework. We will jointly analyze the structures determined in different signaling states to dissect subtle motions that may involve bending, torque, winding/unwinding, or longitudinal sliding of helices. We will also perform mutagenesis and kinase assays to identify the key structural elements responsible for signal coupling between the sensor domains, the helical spine and the effector domain. We will determine the structures and dynamics of full-length photoreceptors by complementary approaches of crystallography and electron microscopy to address whether the structural asymmetry of the sensor and effector domains tethered in the same dimer scaffold plays an important role in allosteric regulation of modular photoreceptors. Our results will not only apply to photoreceptors but will inform the more general principles by which multi-domain signaling proteins such as the more widely studied chemoreceptors detect and process complex environmental signals at the molecular level. Use of light as operands in arithmetic and logic operations that override, negate, or modulate a desired cellular response is of great importance both for basic science and potentially for biotechnology applications.

项目概要 感知和响应复杂环境信号（例如光、氧气和营养物质）的能力 对于生物体的生存和适应至关重要。许多信号蛋白采用多结构域模块化 完成输入信号感知和输出生物反应生成的架构 在同一个蛋白质分子内。基于 bilin 的系统提供了一种广泛使用的模块化系统的示例 光敏色素超家族中的光感受器。由于光很容易穿透细胞膜，因此 可溶性多域光感受器为研究仍然难以捉摸的机制提供了优秀的模型系统 模块化信号蛋白（例如化学感受器）的长程信号传导和变构调节 机械感受器。我们的长期目标是了解模块化感光器如何感知、集成和 在分子水平上转导信号。为了实现这一目标，我们采用生物化学的综合方法， 光谱学、晶体学和冷冻电镜单粒子重建，主要研究动态 晶体学，能够以原子分辨率直接观察结构响应。在这个提案中， 我们使用两个双传感器光感受器来代表两种主要类型的胆碱结合光感受器。两者都是 感觉组氨酸激酶具有不同的颜色感知和响应光的独特信号逻辑 或化学信号。此前，我们已经获得了各种孤立的丰富的结构信息。 静态和动态晶体学领域。在本提案中，我们将研究分子 全长蛋白质中信号整合和变构激活的机制，其中传感器和效应器 域是耦合的。具体来说，我们将通过引入来捕捉每个感觉部位的结构变化 通过配体浸泡和光照射进行扰动。我们将检查结构信号如何 启动以及它们如何通过蛋白质框架传播。我们将共同分析结构 在不同的信号状态下确定，以剖析可能涉及弯曲、扭矩、 卷绕/展开，或螺旋的纵向滑动。我们还将进行诱变和激酶测定 确定负责传感器域之间信号耦合的关键结构元件，螺旋 脊柱和效应器结构域。我们将通过以下方式确定全长光感受器的结构和动力学 晶体学和电子显微镜的互补方法来解决结构是否 束缚在同一二聚体支架中的传感器和效应器结构域的不对称性在 模块化感光器的变构调节。我们的结果不仅适用于光感受器，而且适用于 告知多结构域信号蛋白的更一般原则，例如更广泛研究的 化学感受器在分子水平上检测和处理复杂的环境信号。利用光作为 算术和逻辑运算中覆盖、否定或调节所需细胞反应的操作数是 对于基础科学和生物技术应用潜力都非常重要。