Structures, Dynamics and Signaling Mechanisms of Modular Photoreceptors

项目摘要 感知和响应复杂环境信号的能力，如光、氧和养分 对生物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 实现对输入信号的感知和输出生物响应的生成的体系结构 在相同的蛋白质分子中。BILIN提供了一个广泛使用的模块化系统的例子 光敏色素超家族中的光受体。由于光线可以很容易地穿透细胞膜，这些 可溶性多结构域光感受器为研究青光眼的致病机制提供了良好的模型系统。 化学受体和变构调节等模块化信号蛋白的长程信号和变构调节 机械感受器。我们的长期目标是了解模块化光感受器是如何感知、整合和 在分子水平上传递信号。为了达到这一目标，我们采用了生物化学的综合方法， 光谱学、结晶学和低温EM单粒子重建，重点是动力学 结晶学，能够在原子分辨率下直接观察结构反应。在这份提案中， 我们使用两个双传感器感光器来代表两种主要类型的胆碱结合光感受器。两者都是 以不同的颜色感知和不同的信号逻辑为特征的感觉性组氨酸蛋白对光的反应 或化学信号。此前，我们已经获得了丰富的结构信息，关于各种孤立的 静态和动态的结晶学分析表明，这是一种微区结构。在这个提案中，我们将研究分子 全长蛋白质的信号整合和变构激活机制，其中传感器和效应器 域是耦合的。具体地说，我们将通过引入 通过配体浸泡和光照产生的微扰。我们将研究结构性信号是如何 以及它们是如何通过蛋白质框架传播的。我们将联合分析这些结构 在不同的信号状态下确定，以剖析可能涉及弯曲、扭矩、 螺旋的卷绕/解卷或纵向滑动。我们还将进行突变和激酶检测，以 确定负责传感器域之间的信号耦合的关键结构元件，螺旋 脊椎和效应器域。我们将通过以下方法确定全长光感受器的结构和动力学 结晶学和电子显微镜的互补方法来解决结构是否 在同一个二聚体支架中，传感器和效应器结构域的不对称性在 模块化光感受器的变构调节。我们的结果不仅适用于光感受器，而且将 告知更普遍的原理，通过这些原理，多结构域信号蛋白等得到更广泛的研究 化学感受器在分子水平上检测和处理复杂的环境信号。使用灯光作为 算术和逻辑运算中覆盖、否定或调制所需细胞响应的操作数是 对基础科学和潜在的生物技术应用都非常重要。

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.