EXPLORING LIGHT-SENSING AND SIGNALING MECHANISMS OF BACTERIOPHYTOCHROMES BY C

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 光是原核生物和真核生物的主要环境刺激因子。光敏色素是一种红光感受器，调节植物、真菌和细菌的种子萌发、开花诱导和趋光性等一系列生理过程。植物光敏色素(Phy)和细菌光敏色素(BPH)使用线性四吡咯(BLIN)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等人，2006年)。然而，光转化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，使我们能够确定三种不同的细菌藻色素(来自铜绿假单胞菌的PaBphP和来自沼泽假单胞菌的RpBphP3/RpBphP2)以及几个突变结构在Pr和Pfr状态下的四种不同的光感受域的晶体结构。我们已经解决了任何类型的光敏色素的第一个PFR结构。我们将继续使用静态和时间分辨结晶学以及小角X射线散射技术来探索细菌藻色素中信号转导机制的结构基础。我们在此期间的具体目标是：1.确定具有完整传感器和效应结构域的全长细菌光敏色素(160KD)的晶体结构(静态单色结晶学)；2.在Pr/PFR光转化反应途径中捕获低温陷阱结构中间体(静态单色结晶学)；3.探索常温下光转化过程中的早期结构中间体(在PS-ms时间尺度上)(时间分辨劳厄结晶学)；4.探索将N-末端感受器结构域感受到的光信号传递到C-末端效应器组氨酸激酶域的全球结构变化。这就要求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。

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.