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Structure, Properties and Function of Photoactive Yellow Protein

光活性黄色蛋白的结构、性质和功能

基本信息

批准号：
13480221
负责人：
KATAOKA Mikio
金额：
$ 9.6万
依托单位：
Nara Institute of Science and Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

项目摘要

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.

我们深入研究了光吸收后的结构变化，并寻找了pyp1的靶分子。我们考察了PYP在晶体中的光反应，发现中间产物M在晶体中几乎不可见。这说明了两种可能性：晶体中没有发生反应，或者晶体中的光反应急剧加速。晶体晶格的力约束对光反应有显著影响。基于光照下的CD测量，我们发现M形成导致的结构变化是由n端α-螺旋结构的丢失、c端α-螺旋结构的畸变和β-核的变化组成的。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端区域膨胀，n端区域与c端区域之间的距离增加。这些结构变化是由静电相互作用的变化引起的。7号到15号的区域负责互动。柠檬酸盐与中间体M特异性结合。其中一个结合位点为R52.6。通过对R52Q的晶体结构分析，确定了L形成时控制发色团pKa的必需水分子。从嗜盐异硫螺旋藻细胞中分离出减慢光反应速率的组分。其主要成分是一种蛋白质。净化工作正在进行中。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，以明确功能区和结构区。