Time-Resolved Macromolecular Crystallography

时间分辨高分子晶体学

• 批准号: 8035651
• 负责人: JOHN Keith MOFFAT
• 金额: $ 10.54万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8035651
• 关键词: Address; Architecture; Bacillus subtilis; Bacteria; Behavior; Binding; Biological; Biological Process; Catalysis; Chemicals; Chemoreceptors; Chicago; Collaborations; Coupling; Crystallization; Crystallography; DNA Binding; Electronics; Event; Exhibits; Goals; Guanosine Triphosphate Phosphohydrolases; Kinetics; Knowledge; Life; Ligand Binding; Light; Light Exercise; Link; Malignant Neoplasms; Molecular; Mouse-ear Cress; Oats; Organism; Output; Oxygen; Pathway interactions; Peptide Hydrolases; Photons; Photoreceptors; Plants; Process; Progress Reports; Protein Tyrosine Kinase; Protein-Serine-Threonine Kinases; Proteins; Pseudomonas aeruginosa; Reaction; Resolution; Rhodopseudomonas; Roentgen Rays; Signal Transduction; Signal Transduction Pathway; Site-Directed Mutagenesis; Solutions; Structural Biologist; Structure; System; Techniques; Testing; Thermodynamics; Time; absorption; base; biological systems; chemical kinetics; chemical reaction; chromophore; design; design and construction; fungus; interest; novel; protein-histidine kinase; public health relevance; research study; sensor; success; tool; voltage

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.

描述（由申请人提供）：目前，我们对生物系统中分子水平反应机制的理解主要基于通过高分辨率X射线晶体学和NMR技术获得的静态、化学或物理捕获结构的知识。然而，结构变化的动态方面在所有化学和生物过程中都至关重要，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，我们成功地在光敏系统上进行了~100皮秒时间分辨率和高晶体学分辨率的时间分辨晶体学实验，鉴定了短寿命中间体的结构，并表征了光依赖性的整体机制。这些中间体所占据的信号转导。最近，我们确定了几种基于模块化架构的新型天然信号光感受器的静态晶体结构。那些含有所谓的 LOV 或 BLUF 传感器域的细菌光敏色素对蓝光做出反应，而含有 PAS-GAF-PHY 域的细菌光敏色素则对红光/远红光做出反应。我们现在将对这些蛋白质以及包含传感器和效应器（输出）域的较长结构进行时间分辨晶体学实验。我们解决以下问题：信号是如何生成和传输的？光如何控制活动？在每种情况下，光依赖性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，但我们相信信号转导的一般原理是存在的。事实上，我们已经制备了嵌合的人工光感受器，其中我们使光敏感而具有通常光惰性的生物活性，例如光感受器。组氨酸激酶或 DNA 结合。我们将探索这些人造光感受器来测试和扩展我们的一般原理，提供有用的工具并提供新的结晶目标。最后，人造光感受器克服了时间分辨晶体学的限制：许多有趣的系统不依赖于光。我们将探讨这个问题：这些方法有多普遍？公共健康相关性：许多癌症与配体与化学感受器结合驱动的信号转导途径紊乱有关。这些途径可能是已知的，但分子水平的机制却未知。我们研究的天然和人造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：两者的信号转导的热力学和结构原理可能相似。