Abstract：
Cyanobacteriochromes (CBCRs) are phytochrome-related photosensory proteins that play an essential role in regulating phototaxis, chromatic acclimation, and cell aggregation in cyanobacteria. Here, we apply solid-state NMR spectroscopy to the red/green GAF2 domain of the CBCR AnPixJ assembled in vitro with a uniformly 13C- and 15N-labeled bilin chromophore, tracking changes in electronic structure, geometry, and structural heterogeneity of the chromophore as well as intimate contacts between the chromophore and protein residues in the photocycle. Our data confirm that the bilin ring D is strongly twisted with respect to the B-C plane in both dark and photoproduct states. We also identify a greater structural heterogeneity of the bilin chromophore in the photoproduct than in the dark state. In addition, the binding pocket is more hydrated in the photoproduct. Observation of interfacial 1H contacts of the photoproduct chromophore, together with quantum mechanics/molecular mechanics (QM/MM)-based structural models for this photoproduct, clearly suggests the presence of a biprotonated (cationic) imidazolium side-chain for a conserved histidine residue (322) at a distance of ~2.7 Å, generalizing the recent theoretical findings that explicitly link the structural heterogeneity of the dark-state chromophore to the protonation of this specific residue. Moreover, we examine pH effects on this in vitro assembled holoprotein, showing a substantially altered electronic structure and protonation of the photoproduct chromophore even with a small pH drop from 7.8 to 7.2. Our studies provide further information regarding the light- and pH-induced changes of the chromophore and the rearrangements of the hydrogen-bonding and electrostatic interaction network around it. Possible correlations between structural heterogeneity of the chromophore, protonation of the histidine residue nearby, and hydration of the pocket in both photostates are discussed.
摘要：
蓝藻色素（CBCR）是植物色素相关的光感蛋白，在调节趋光性中起着至关重要的作用。色彩适应，和蓝细菌中的细胞聚集。这里,我们将固态NMR光谱应用于CBCRAnPixJ的红色/绿色GAF2域，该域在体外与均匀的13C-和15N-标记的bilin发色团组装，跟踪电子结构的变化，几何图形,和生色团的结构异质性以及生色团和光循环中蛋白质残基之间的密切接触。我们的数据证实，在黑暗和光产物状态下，bilin环D相对于B-C平面强烈扭曲。我们还发现，与黑暗状态相比，光产物中的bilin发色团的结构异质性更大。此外,结合袋在光产品中更水合。观察光产物发色团的界面1H接触，结合基于量子力学/分子力学(QM/MM)的结构模型，清楚地表明，在〜2.7的距离处存在保守的组氨酸残基（322）的双质子化（阳离子）咪唑鎓侧链，概括了最近的理论发现，这些发现明确地将暗态发色团的结构异质性与该特定残基的质子化联系起来。此外，我们研究了pH对这种体外组装的全蛋白的影响，显示光产物发色团的基本改变的电子结构和质子化,即使pH从7.8小到7.2。我们的研究提供了有关光和pH引起的生色团变化以及周围氢键和静电相互作用网络重排的进一步信息。生色团的结构异质性之间可能的相关性，附近组氨酸残基的质子化，并讨论了两种光状态下口袋的水合作用。
