Time-Resolved Macromolecular Crystallography

时间分辨高分子晶体学

• 批准号: 7756665
• 负责人: JOHN Keith MOFFAT
• 金额: $ 60.23万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-09-01 至 2012-12-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7756665
• 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）：我们对分子水平生物系统中反应机制的理解目前主要基于对高分辨率X射线晶体学和NMR技术获得的静态，化学或物理捕获的结构的知识。然而，结构变化的动态方面在所有化学和生物学过程中至关重要，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，我们成功地进行了时间分辨的晶体学实验，并在光敏系统上使用了〜100 picsecond Time分辨率和高晶体学分辨率，并确定了短寿命中间体的结构，并表征了这些Intermediedipiatiatiatiatiatiate的光依赖性信号传输的总体机制。最近，我们确定了基于模块化体系结构的几种新型，天然的信号感光体的静态晶体结构。那些包含所谓的LOV或BLUF传感器域对蓝光响应，以及含有PAS-GAF-PHY域的拟菌卵形型，以红色/远红色的光。现在，我们将对这些蛋白质进行时间分辨的晶体学实验，并在包含传感器和效应器（输出）域的更长构建体上进行。我们解决了问题：信号如何生成和传输？活动如何由光控制？在每个中，光依赖性信号转导的机制是什么？尽管天然感光体的化学和结构多样性很高，但我们认为信号转导的一般原理存在。实际上，我们已经准备了嵌合，人造感光体，其中使光敏感应敏感为正常的轻度启动生物学活性，例如组氨酸激酶或DNA结合。我们将探索这些人工感受器，以测试和扩展我们的一般原则，提供有用的工具并为结晶提供新的目标。最后，人工感受器克服了时间分辨晶体学的局限性：许多有趣的系统不是光依赖的。我们将提出一个问题：这些方法有多通用？公共卫生相关性：许多癌症与配体与化学感受器的结合驱动的信号转导途径的危险有关。这些途径可能是已知的，但分子水平的机制却不知道。我们研究的分子机制的天然和人工感受器与化学感受器具有相似之处，但也具有关键的差异：信号转导的热力学和结构原理可能相似。