Time-Resolved Macromolecular Crystallography

时间分辨高分子晶体学

• 批准号: 7006978
• 负责人: JOHN Keith MOFFAT
• 金额: $ 54.84万
• 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 2008-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7006978
• 关键词: X ray crystallography; bioimaging /biomedical imaging; carboxyhemoglobin; chemical binding; chemical kinetics; conformation; heme; hemoglobin; lasers; mathematical model; method development; molecular dynamics; myoglobin; nonvisual photoreceptor; pancreatic ribonuclease; photoactivation; photochemistry; photolysis; protein structure function; rhodopsin; structural biology; temperature; thermodynamics; time resolved data

DESCRIPTION (provided by applicant): Our understanding of reaction mechanisms in biological systems at the molecular level is presently based almost entirely on knowledge of static, chemically- or physically-trapped structures. However, the dynamic aspects of changes in structure are critical during processes such as catalysis, ligand binding and release, protein (re)folding and signal transduction. These changes can be extremely rapid and the lifetime of structural intermediates correspondingly small, from seconds to picoseconds and even shorter. The changes can also be small in spatial extent, which demands high crystallographic resolution and accurate imeasurement of small changes in X-ray structure amplitudes. We have successfully conducted high resolution, time-resolved crystallographic experiments with approximately 100 picosecond time resolution on light-sensitive systems, identified the structures of short-lived intermediates and characterized the overall mechanism. We will apply time-resolved crystallography to elucidate the mechanism of light-dependent signal transduction in selected, water-soluble photoreceptors, at the atomic level. These contain one or more sensor domains that bind a small, non-protein chromophore, together with an effector domain. After absorption of a photon by the chromophore, prompt electronic and structural changes in the chromophore itself cause structural changes in the sensor domain and ultimately in the effector domain. These changes in structure modulate the activity of the effector domain; an otherwise light-inert biochemical activity such as phosphorylation becomes light-sensitive. The photoreceptor sensor domains bind chemically diverse chromophores such as FMN, FAD, p-coumaric acid or heme and are linked to structurally diverse effector domains with; e.g., serine/threonine or histidine kinase activity. Bacterial and plant photoreceptor systems will be studied, as isolated sensor and effector domains and in naturally-occurring, covalent and noncovalent complexes.

描述（由申请人提供）：我们对生物系统中分子水平反应机制的理解目前几乎完全基于静态、化学或物理捕获结构的知识。然而，结构变化的动态方面在催化、配体结合和释放、蛋白质（重新）折叠和信号转导等过程中至关重要。这些变化可能非常迅速，结构中间体的寿命相应地很短，从几秒到皮秒甚至更短。这些变化在空间范围内也可能很小，这需要高晶体学分辨率和精确测量 X 射线结构振幅的微小变化。我们已经成功地在光敏系统上进行了大约 100 皮秒时间分辨率的高分辨率、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整体机制。 我们将应用时间分辨晶体学在原子水平上阐明选定的水溶性光感受器中光依赖性信号转导的机制。它们包含一个或多个与小型非蛋白质发色团结合的传感器结构域以及效应结构域。发色团吸收光子后，发色团本身的迅速电子和结构变化导致传感器域并最终引起效应器域的结构变化。这些结构变化调节效应域的活性；原本对光惰性的生化活性（例如磷酸化）变得对光敏感。光感受器传感器域结合化学上多样化的发色团，例如 FMN、FAD、对香豆酸或血红素，并通过以下方式与结构上多样化的效应器域连接：例如，丝氨酸/苏氨酸或组氨酸激酶活性。将研究细菌和植物光感受器系统，作为分离的传感器和效应器域以及天然存在的共价和非共价复合物。