Time-Resolved Macromolecular Crystallography

描述(申请人提供)：我们目前在分子水平上对生物体系中的反应机理的了解主要基于通过高分辨率X射线结晶学和核磁共振技术获得的静态、化学或物理陷阱结构的知识。然而，结构变化的动态方面在所有的化学和生物过程中都是至关重要的，例如催化、配体结合和释放以及信号转导。为了探索信号转导的机制，我们成功地在光敏体系上进行了时间分辨实验，时间分辨率为~100皮秒，晶体分辨率高，确定了短寿命中间体的结构，并表征了这些中间体所存在的光依赖信号转导的整体机制。最近，我们已经确定了几种新型的、自然发生的、基于模块化结构的信号光感受器的静态晶体结构。那些含有LOV或BLUF传感器结构域的细菌对蓝光有反应，而含有PAS-GAF-PHY结构域的细菌光敏蛋白对红色/远红光有反应。我们现在将对这些蛋白质以及包含传感器和效应器(输出)结构域的较长结构进行时间分辨结晶学实验。我们解决了这些问题：信号是如何产生和传输的？光是如何控制活动的？其中，光依赖信号转导的机制是什么？尽管天然光感受器具有高度的化学和结构多样性，但我们认为信号转导的一般原理是存在的。事实上，我们已经准备了嵌合的人造光感受器，在其中我们已经使光敏感通常具有光惰性的生物活性，例如组氨酸激酶或DNA结合。我们将探索这些人工光感受器，以测试和扩展我们的一般原理，提供有用的工具，并为结晶提供新的靶点。最后，人工光感受器克服了时间分辨结晶学的局限性：许多有趣的系统不依赖于光。我们将追问这个问题：这些方法有多普遍？公共卫生相关性：许多癌症与信号转导通路的紊乱有关，由与化学感受器结合的配体驱动。这些途径可能是已知的，但分子水平上的机制尚不清楚。我们研究的天然和人工光感受器的分子机制与化学感受器有相似之处，但也有关键区别：信号转导的热力学和结构原理可能在两者中相似。

DESCRIPTION (provided by applicant): Our understanding of reaction mechanisms in biological systems at the molecular level is presently based largely on knowledge of static, chemically- or physically-trapped structures obtained by high resolution X-ray crystallographic and NMR techniques. However, the dynamic aspects of changes in structure are critical in all chemical and biological processes, for example catalysis, ligand binding and release, and signal transduction. To explore the mechanisms of signal transduction, we have successfully conducted time-resolved crystallographic experiments with ~100 picosecond time resolution and high crystallographic resolution on light-sensitive systems, identified the structures of short-lived intermediates and characterized the overall mechanism of light-dependent signal transduction which these intermediates populate. Recently, we have determined the static crystal structures of several novel, naturally-occurring, signaling photoreceptors which are based on a modular architecture. Those containing so-called LOV or BLUF sensor domains respond to blue light, and bacteriophytochromes containing PAS-GAF-PHY domains to red/far-red light. We will now conduct time-resolved crystallographic experiments on these proteins, and on longer constructs that contain both sensor and effector (output) domains. We address the questions: How is a signal generated and transmitted? How is activity controlled by light? In each, what is the mechanism of light-dependent signal transduction? Despite the high chemical and structural diversity in natural photoreceptors, we believe that general principles of signal transduction exist. Indeed, we have prepared chimeric, artificial photoreceptors in which we have made light-sensitive a normally light-inert biological activity e.g. histidine kinase or DNA binding. We will explore these artificial photoreceptors to test and expand our general principles, afford useful tools and offer new targets for crystallization. Finally, artificial photoreceptors overcome a limitation of time-resolved crystallography: many interesting systems are not light-dependent. We will pursue the question: How general are these approaches? PUBLIC HEALTH RELEVANCE: Many cancers are associated with derangement of signal transduction pathways, driven by ligand binding to chemoreceptors. The pathways may be known but the mechanisms at the molecular level are not. The natural and artificial photoreceptors whose molecular mechanisms we study have parallels to, but also key differences from, chemoreceptors: the thermodynamic and structural principles of signal transduction are likely to be similar in both.