Functionally-important protein dynamics of photosensor proteins as revealed by time-resolved resonance Raman spectroscopy

利用时间分辨共振拉曼光谱研究了一些传感器蛋白的重要功能蛋白动力学。主要结果如下。(1)构建时间分辨紫外共振拉曼光谱仪(2)皮秒蛋白对光活性黄蛋白（PIP）发色团异构化的响应测定PYP的皮秒时间分辨紫外共振拉曼光谱（UVRR）。由于酪氨酸和色氨酸残基的振动引起的UVRR波段在光反应中表现出光谱变化。发现在pG*态下，发色团与Y42之间的氢键强度增加。色氨酸带的超快变化表明，光诱导的发色团结构变化在皮秒内传播到距离发色团12a的W119区域。FixLFixL是一种基于血红素的O2传感器蛋白，它通过激活转录激活子FixJ来响应低O2浓度。信号转导是由O2从FixL的传感器结构域解离开始的，导致蛋白质构象变化，并传递到组氨酸激酶结构域。为了深入了解FixL的传感机制，我们使用时间分辨共振拉曼光谱在皮秒到毫秒的时间范围内监测了配体解离后蛋白质结构的变化。FixL光谱分析表明，动态结构变化分为三个阶段。在第一步观察到与配体相关的结构动力学。在比较这些结构变化的基础上，提出了FixL的信号转导方案。在传感器结构域和全长蛋白中都观察到类似的光谱变化，尽管前者的结构变化比后者更快。这种速率的差异表明，发生在血红素口袋中的结构变化与激酶结构域的变化是耦合的。少

Functionally-important protein dynamics of some sensor proteins have been studied by time-resolved resonance Raman spectroscopy. Chief results are as follows.(1) Construction of a time-resolved ultraviolet resonance Raman spectrometer(2) Picosecond Protein Response to the Chromophore Isomerization of Photoactive Yellow Protein (PIP)Picosecond time-resolved ultraviolet resonance Raman (UVRR) spectra of PYP were measured. UVRR bands attributed to the vibration of tyrosine and tryptophan residues showed a spectral change upon photoreaction. It was found that the hydrogen-bond strength between the chromophore and Y42 increases in the pG* state. The ultrafast change in the tryptophan band revealed that a photo-induced structural change of the chromophore had propagated to the W119 region, located 12 A from the chromophore, within picoseconds.(3) Structural Dynamics of FixLon Signal Transduction and Ligand Discrimination FixLFixL is a heme-based O2 sensor protein, which responds to low O2 co … More ncentration by activating the transcriptional activator FixJ. Signal transduction is initiated by the dissociation of O2 from the sensor domain of FixL, resulting in protein conformational changes that are transmitted to a histidine kinase domain. To gain insight into the FixL sensing mechanism, we monitored changes in the protein's structure in the picosecond to millisecond timeframe, following the dissociation of the ligand using time-resolved resonance Raman spectroscopy. The FixL spectra show that there are three steps in the dynamic structural changes. Ligand-dependent structural dynamics are observed in the earliest step. Based on comparisons of these structural changes, a scheme for the signal transduction of FixL is proposed. Similar spectral changes were observed both for the sensor domain and for the full length protein, although structural changes occurred faster with the former than with the latter. This difference in rate suggests that the structural changes occurring in the heme pocket are coupled to those of the kinase domain. Less