EXPLORING LIGHT-SENSING AND SIGNALING MECHANISMS OF BACTERIOPHYTOCHROMES BY C

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 光是原核生物和真核生物的主要环境刺激。 光敏色素是一类红光光感受器，在植物种子萌发、成花诱导和趋光性等生理过程中起重要作用。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，并在红色吸收（Pr）和远红色吸收（Pfr）状态之间进行光转换（Rockwell等人2006）。然而，光转化和信号转导机制的分子和机制细节仍然模糊。在最后一个提案（#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）和Pr和Pfr状态下的几种突变结构。我们已经解决了任何种类光敏色素的第一个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。 我们在这段时间的具体目标是：1。确定具有完整传感器和效应器结构域的全长细菌光敏色素（160 KD）的晶体结构（静态单色晶体学）; 2.在Pr/Pfr光转换反应途径中低温捕获结构中间体（静态单色晶体学）; 3.探测在环境温度下光转化期间的早期结构中间体（在ps-ms时间尺度中）（时间分辨劳厄晶体学）; 4.探索将N-末端感觉域感知的光信号传递到C-末端效应器组氨酸激酶域的全局结构变化。这需要SAXS研究溶液中的样品，因为这种全局结构重排通常局限于晶格中。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.