Structure, Properties and Function of Photoactive Yellow Protein

我们深入研究了光吸收引起的结构变化，寻找了PYP的目标分子。1.我们考察了PYP在晶体中的光反应，发现M中间体在晶体中很难观察到。这暗示了两种可能性：晶体中不发生反应或晶体中的光反应极其加速。2.基于光照下的CD测量，我们揭示了由于M形成引起的结构变化包括N-末端区域α-螺旋结构的丧失、C-末端结构域α-螺旋的扭曲和β-核心的变化。3.我们还通过光照下的溶液X射线散射发现，随着M中间体的形成，C-末端结构域膨胀，N-末端区域与C-末端结构域之间的距离增加。4.这些结构变化是由静电相互作用的变化引起的。从7号到15号的区域负责相互作用。柠檬酸离子与M中间体特异结合。其中一个结合部位是R52.6。通过R52Q的晶体结构分析，确定了控制L组发色团pKa的主要水分子。7.从盐生螺旋藻细胞外分离出减缓光反应速率的组分。主要成分被发现是一种蛋白质。8.设计了具有简化序列的人工PYP，并在大肠杆菌中进行了表达和纯化。对这些简化的PYP的结构、性质和光反应进行了研究，以明确其功能区和结构区。

We have studied intensively the structural changes upon light absorption, and searched the target molecule of PYP.1.We examined the photoreaction of PYP in a crystal, and found that the M intermediate is hardly observable in a crystal. This suggests two possibilities : no reaction is occurred in a crystal or the photoreaction accelerates extremely in a crystal. The photoreaction is significantly affected from the force constraint of crystalline lattice.2.Based on the CD measurements under illumination, we revealed that the structural changes due to M formation are composed of the loss of α-helical structure of the N-terminal region, the distortion of α-helix of C-terminal domain and the changes in β-core.3.We also revealed by solution X-ray scattering under Illumination that the C-terminal domain swells and the distance between the N-terminal region and the C-terminal domain increases upon the formation of the M intermediate.4.These structural changes are induced by the changes in the electrostatic interaction. The region from the 7th and the 15th is responsible to the Interaction.Citrate ion binds specifically to the M intermediate. One of the binding sites is found to be R52.6.Through the crystal structural analysis of R52Q, the essential water molecule that controls the pKa of the chromophore at the L formation is identified.7.The fraction that slows down the photoreaction rate was isolated from Ectothiorhodospira halophila cell. The major component is found to be a protein. The purification is now under way.8.Artificial PYP's with the simplified sequences are designed, expressed in E.coli and purified. The structures, properties and photoreactions of these simplified PYP's were examined to clarify the functional regions and structural regions.