Studies on the Photoreactions and Tertiary Structure of Yellow Protein by Use of the Site-Directed Mutagenesis

光活性黄蛋白(PYP)是一种在紫色光合作用细菌嗜盐异硫杆菌中发现的水溶性光感受器蛋白，由于其三级结构已被X射线结晶学和核磁共振所阐明，因此被认为是从原子水平上理解光接收和信号转导机制的最合适的靶标之一。PYP与Tyr-42、Glu-46和Thr5O形成氢键网络，并被Arg-52稳定。制备了这些氨基酸被取代的突变体，并研究了它们的M中间体的寿命。Y42A和E46Q的M中间体的衰变速度快于野生型，而E46A、T50A、T5OV和R52Q的M中间体的衰变较慢，这表明不仅氢键的强度而且大部分残基对M中间体的行为起调节作用。用ETIR研究了发色团周围氢键网络的变化，发现在B、H和L中间体中，Glu-46的氢键比PYP强，而在M中间体中，Glu-46被质子化，这表明M中间体中PYP的三级结构发生了显著的变化。在反式对香豆酸的照射下，确定了顺式对香豆酸的振动模式特征，并分析了PYP及其中间体的FTIR光谱差异，发现所有中间体都有顺式振动模式的特征，但PYP中没有顺式模式的振动模式，这表明PYP中的主要光化学事件是类生色团视网膜蛋白的异构化。

Photoactive yellow protein (PYP) is a water-soluble photoreceptor protein found in the purple photosynthetic bacterium, Ectothiorhodospira halophila.Because its tertiary structure has been elucidated by X-ray crystallography and NMR, it is considered to be one of the most suitable target to understand the mechanisms of photoreception and signal transduction in the atomic level.In this project, the photoreaction of PYP was studied using the PYP mutants prepared by the methods we developed.1. The chromophore of PYP together with Tyr-42, Glu-46 and Thr5O forms the hydrogen-bonding network, which is stabilized by Arg-52.The mutants in which these amino acids were replaced were prepared and the lifetimes of their M intermediates were studied.The M intermediates of Y42A and E46Q decayed faster than that of wild type whereas those of E46A, T5OA, T5OV and R52Q decayed slower, indicating that not only the strength of the hydrogen-bond but also the bulk of the residues regulates the behavior of the M intermediate.2. Changes in the hydrogen bonding network around the chromophore was studied by ETIR.The hydrogen-bond of Glu-46 was stronger in B, H, and L intermediates than that of PYP, whereas Glu-46 was protonated in M intermediate.It suggests that the tertiary structure of PYP is remarkably altered in M intermediate.3. The vibrational modes characteristic for cis-p-coumaric acid were identified by irradiation of trans-p-coumaric acid.Then the difference FTIR spectra between PYP and its intermediates were analyzed.The vibrational modes characteristic for cis form were found in all the intermediates but they were not in PYP.It indicates that the primary photochemical event in PYP is the isomerization of the chromophore like retinal proteins.