EXPLORING LIGHT-SENSING AND SIGNALING MECHANISMS OF BACTERIOPHYTOCHROMES BY C

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH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。 光敏色素是一类红光光感受器，在植物、真菌和细菌的种子萌发、成花诱导和趋光性等生理过程中起着重要的调控作用。植物光敏色素（Phy）和细菌光敏色素（Bph）使用线性四吡咯（胆色素）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 et al，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不清。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，这些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来研究溶液中的样品，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. 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.