EXPLORING LIGHT-SENSING AND SIGNALING MECHANISMS OF BACTERIOPHYTOCHROMES BY C

利用 C 探索细菌植物色素的光传感和信号传导机制

• 批准号: 8171982
• 负责人: JOHN Keith MOFFAT
• 金额: $ 4.01万
• 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: 2010-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8171982
• 关键词: Bacteria; Bilin; C-terminal; Computer Retrieval of Information on Scientific Projects Database; Crystallography; DNA Sequence Rearrangement; Data; Eukaryota; Funding; Germination; Grant; Institution; Length; Light; Molecular; N-terminal; Pathway interactions; Photoreceptors; Physiological Processes; Phytochrome; Plants; Prokaryotic Cells; Pseudomonas aeruginosa; Reaction; Research; Research Personnel; Resources; Roentgen Rays; Sampling Studies; Seeds; Sensory; Signal Transduction; Solutions; Source; Stimulus; Structure; Techniques; Temperature; Tetrapyrroles; Time; United States National Institutes of Health; base; chromophore; mutant; plant fungi; protein-histidine kinase; research study; sensor

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Light is a major environmental stimulus for both prokaryotes and eukaryotes. Phytochromes are red light photoreceptors that regulate a wide range of physiological processes such as seed germination, floral induction and phototaxis in plants, fungi and bacteria. Plant phytochromes (Phy) and bacteriophytochromes (Bph) use a linear tetrapyrrole (bilin) as a chromophore and photo-convert between red-absorbing (Pr) and far-red-absorbing (Pfr) states (Rockwell et al, 2006). However, molecular and mechanistic details of photoconversion and signal transduction mechanisms remain obscure. During the last proposal (#6457) period, we have successfully conducted experiments and collected data that allow us determine four different crystal structures of the photosensory domains from three distinct bacteriophytochromes (PaBphP from P. aeruginosa and RpBphP3/RpBphP2 from R. palustris) and several mutant structures in both Pr and Pfr states. We have solved the very first Pfr structure for phytochromes of any kind. We will continue to explore structural basis of signal transduction mechanisms in bacteriophytochromes using static and time-resolved crystallography as well as small-angle X-ray scattering techniques. Our specific aims during this proposal period are: 1. to determine crystal structures of full-length bacteriophytochromes (160KD) with intact sensor and effector domains (static monochromatic crystallography); 2. to cryo-trap structural intermediates during Pr/Pfr photoconversion reaction pathways (static monochromatic crystallography); 3. to probe early structural intermediates (in ps-ms time scale) during photoconversion at ambient temperature (time-resolved Laue crystallography); 4. to explore global structural changes that transmit light signal perceived by the N-terminal sensory domains to the C-terminal effector histidine kinase domain. This requires SAXS to study samples in solution, since such global structural rearrangements are often limited in crystal lattice.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 中心，不一定是研究者的机构。 光是原核生物和真核生物的主要环境刺激物。 光敏色素是红光光感受器，调节多种生理过程，例如植物、真菌和细菌的种子发芽、成花诱导和趋光性。植物光敏色素 (Phy) 和细菌光敏色素 (Bph) 使用线性四吡咯 (bilin) 作为发色团，并在红光吸收 (Pr) 和远红光吸收 (Pfr) 状态之间进行光转换（Rockwell 等，2006）。然而，光转换和信号转导机制的分子和机制细节仍然不清楚。在上一个提案 (#6457) 期间，我们成功地进行了实验并收集了数据，使我们能够确定来自三种不同细菌光敏色素（来自铜绿假单胞菌的 PaBphP 和来自 R. palustris 的 RpBphP3/RpBphP2）的光感域的四种不同晶体结构，以及Pr 和 Pfr 状态下的几种突变结构。我们已经解决了任何类型光敏色素的第一个 Pfr 结构。我们将继续利用静态和时间分辨晶体学以及小角度X射线散射技术探索细菌光敏色素信号转导机制的结构基础。 我们在此提案期间的具体目标是： 1. 确定具有完整传感器和效应域的全长细菌光敏色素（160KD）的晶体结构（静态单色晶体学）； 2. 在 Pr/Pfr 光转换反应途径中冷冻捕获结构中间体（静态单色晶体学）； 3. 探测环境温度下光转换过程中的早期结构中间体（以 ps-ms 时间尺度）（时间分辨劳厄晶体学）； 4. 探索将 N 端感觉域感知的光信号传递到 C 端效应器组氨酸激酶域的整体结构变化。这需要 SAXS 研究溶液中的样品，因为这种全局结构重排通常仅限于晶格。