PHYTOCHROME SIGNAL TRANSDUCTION - BLUE ABSORBING CYANOBACTERIAL PHYTOCHROMES

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 光敏色素(Phys)是一组不同的光感受器，通过结合血红素衍生的线性四氢吡咯成为光致变色来调节植物和细菌中的一系列重要过程。正则物理由两个光谱态组成，一个是红光吸收基态(Pr)，一个是远红光激发态(Pfr)。最近的基因组和生化分析揭示了蓝藻所特有的一组不同的类Phy蓝绿色光致变色蛋白，蓝藻碘变色蛋白(CBCs)。与典型的吸收红色的蓝藻Phys不同，CBCs依赖于一个半胱氨酸来附着藻蓝胆素(PCB)发色团，而CBCs依赖于第二个保守的半胱氨酸，该半胱氨酸使其标志性的蓝绿(PbPG)光可逆特性得以实现，可能是通过缩短PCBp共轭体系。以长形嗜热菌BP-1蛋白tll0569(TePixJ-GAF)的光致变色结构域染色肽(TePixJ-GAF)为模型CBC，通过诱变证明第二个Cys(Cys-494)确实与BLIN形成了共价键。吸光度、IR、核磁共振和RR光谱技术表明，额外的Cys494-PCB加合物很可能发生在四吡咯的A-B亚甲基桥上，产生更多去共轭的藻红素样四吡咯。这种连接在整个光循环中保持不变，所有四个多氯联苯氮在铅和PG状态下都被质子化。用均匀同位素标记的15N-TePixJ-GAF多肽的核磁共振谱表明，Pb态和Pg态可能保持为两个完全不同的结构，没有暗回复或交叉污染的证据。我们将尝试确定含有发色团的TePixJ-GAF结构域的结构。这个构造物可以光转换，只有~20 kDa；我们还想测定它在蓝/绿光照射下的铅和PG的形态。

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. Phytochromes (Phys) are a diverse collection of photoreceptors that regulate an array of important processes in plants and bacteria by binding heme-derived linear tetrapyrroles to become photochromic. The canonical Phys consist of two spectral states, a red-absorbing ground state (Pr) and a far red-absorbing excited state (Pfr). Recent genomic and biochemical analyses have revealed a divergent subset of Phy-like blue-green photochromic proteins unique to cyanobacteria, the cyanobacteriochromes (Cbcs). Unlike canonical red-absorbing cyanobacterial Phys, which rely on a single cysteine for attachment of the phycocyanobilin (PCB) chromophore, Cbcs rely on a second conserved cysteine that enables their hallmark blue-green (Pb-Pg) photoreversible properties, presumably by shortening the p conjugation system of PCB. Using the photochromic GAF domain chromopeptide from Thermosynechococcus elongatus BP-1 protein tll0569 (TePixJ-GAF) as a model Cbc, we demonstrated by mutagenesis that the second Cys (Cys-494) indeed forms a covalent linkage to the bilin. Absorbance, IR, NMR, and RR spectroscopic techniques show that the additional Cys494-PCB adduct most likely occurs at the tetrapyrrole A-B methine bridge, yielding a more de-conjugated phycoviolobilin-like tetrapyrrole. This linkage remains intact throughout the photocycle, with all four PCB nitrogens being protonated in both Pb and Pg states. NMR spectroscopy with uniformly isotopically-labeled 15N-TePixJ-GAF polypeptide revealed that Pb and Pg states may be maintained as two entirely distinct structures without evidence of dark reversion nor cross contamination. We will attempt structure determination of the TePixJ-GAF domain which houses the chromophore. The construct photoconverts and is only ~20 kDa; we would like to also determine its Pb andf Pg forms upon blue/green light exposure.