Structure, Properties and Function of Photoactive Yellow Protein

1.研究了PYP在晶体中的光反应，发现在晶体中几乎观察不到M中间体。这表明了两种可能性：在晶体中不发生反应或在晶体中光反应极度加速。光反应受到晶格力约束的显着影响。2.基于光照下的CD测量，我们揭示了M形成引起的结构变化包括N端区域α螺旋结构的丧失，C-末端结构域的α-螺旋变形和β-核心的变化. 3.我们还通过溶液X-射线散射在光照下揭示了C-末端结构域的C-随着M中间体的形成，N-末端区域和C-末端区域之间的距离增加，末端结构域膨胀。4.这些结构变化是由静电相互作用的变化引起的。第7和第15位的区域负责相互作用，柠檬酸根离子特异性地与M中间体结合。6.通过对R52 Q的晶体结构分析，确定了在L形成过程中控制发色团pKa的主要水分子。7.从嗜盐外硫红螺旋菌（Ectothiorhodospira halophila）细胞中分离到一个减缓光反应速率的组分。发现主要成分是蛋白质。8.设计了具有简化序列的人工PYP，在大肠杆菌中表达并纯化。对这些简化的PYP的结构、性质和光反应进行了研究，以阐明其功能区和结构区。

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.