PHYTOCHROME SIGNAL TRANSDUCTION - BLUE ABSORBING CYANOBACTERIAL PHYTOCHROMES

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。 光敏色素（Phytochromes，Phys）是一类光感受器，通过与血红素衍生的线性四吡咯结合而产生光致变色，从而调节植物和细菌中的一系列重要过程。 正则Phys由两个光谱态组成，一个吸收红光的基态（Pr）和一个吸收远红光的激发态（Pfr）。 最近的基因组和生物化学分析揭示了一个不同的子集的Phy样蓝绿色光致变色蛋白独特的蓝藻，蓝藻色素（Cbcs）。 与典型的红色吸收蓝藻Phys不同，其依赖于单个半胱氨酸来附着藻蓝胆素（PCB）发色团，Cbcs依赖于第二个保守的半胱氨酸，这使得其标志性的蓝绿色（Pb-Pg）光可逆特性，可能是通过缩短PCB的p共轭系统。 使用来自细长热聚球藻BP-1蛋白tll 0569（TePixJ-GAF）的光致变色GAF结构域色肽作为Cbc模型，我们通过诱变证明第二个Cys（Cys-494）确实与胆色素形成共价键。 吸光度，IR，NMR和RR光谱技术表明，额外的Cys 494-PCB加合物最有可能发生在四吡咯A-B次甲基桥，产生更去共轭藻紫胆素样四吡咯。 这种联系在整个光循环中保持完整，所有四个PCB氮在Pb和Pg状态下都被质子化。 用均匀同位素标记的15 N-TePixJ-GAF多肽的NMR光谱显示，Pb和Pg状态可以保持为两种完全不同的结构，而没有暗回复或交叉污染的证据。我们将尝试结构测定的TePixJ-GAF结构域的房屋发色团。该构建体光转换并且仅为~20 kDa;我们还想确定其在蓝/绿色光暴露后的Pb和f Pg形式。

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.