Structures, Dynamics and Signaling Mechanisms of Modular Photoreceptors

项目摘要 感知和响应复杂环境信号（如光、氧气和营养物质）的能力， 对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结构， 实现输入信号的感知和输出生物响应的生成的体系结构 在同一个蛋白质分子中。广泛使用的模块化系统的一个例子是由基于bilin的 光敏色素超家族中的光感受器。由于光可以很容易地穿透细胞膜， 可溶性多域光感受器为研究仍然难以捉摸的光感受器的机制提供了极好的模型系统。 在模块化信号蛋白如化学感受器和 机械感受器我们的长期目标是了解模块化光感受器如何感知，整合， 在分子水平上发出信号。为了实现这一目标，我们采用了生物化学的综合方法， 光谱学，晶体学和cryoEM单粒子重建，主要致力于动态 晶体学，其能够以原子分辨率直接观察结构响应。在这一提议中， 我们使用两种双传感器光感受器来代表两种主要类型的胆色素结合光感受器。两者都是 感觉组氨酸激酶，其特征在于不同的颜色感知和对光的响应的不同信号逻辑 或化学信号。以前，我们已经获得了各种分离的结构信息， 域的静态和动态晶体学。在这个建议中，我们将研究分子 在全长蛋白质中的信号整合和变构激活的机制，其中传感器和效应器 域耦合。具体来说，我们将通过引入 通过配体浸泡和光照进行扰动。我们将研究结构信号是如何 以及它们如何通过蛋白质框架传播。我们将共同分析 在不同的信号状态下确定，以剖析可能涉及弯曲，扭矩， 卷绕/退绕或螺旋的纵向滑动。我们还将进行诱变和激酶测定， 识别负责传感器域之间信号耦合的关键结构元件，螺旋 棘和效应域。我们将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，以解决是否结构 拴系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 模块光感受器的变构调节。我们的结果不仅适用于光感受器， 告知多结构域信号蛋白，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 算术和逻辑运算中的操作数可以覆盖、否定或调节所需的细胞反应， 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.