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

用时间分辨共振拉曼光谱研究了一些传感器蛋白的功能重要蛋白质动力学。主要结果如下：(1)时间分辨紫外共振拉曼光谱的建立(2)测量了PYP的皮秒蛋白质对光活性黄蛋白(PIP)发色团异构化的时间分辨紫外共振拉曼(UVRR)光谱。由于酪氨酸和色氨酸残基的振动，UVRR谱带在光反应过程中发生了光谱变化。研究发现，在PG*态下，发色团与Y42之间的氢键强度增大。色氨酸带的超快变化表明，光致发色团的结构变化在皮秒内传播到距离发色团12 A的W119区域。(3)FixLon信号转导和配基识别FixL的结构动力学FixL是一种基于血红素的O2感受器蛋白，对低O2 co…做出反应通过激活转录激活因子FixJ获得更高的浓度。信号转导是由O2从FixL的感应域解离而开始的，导致蛋白质构象变化，这些变化被传递到组氨酸激酶域。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监测了配体解离后蛋白质结构在皮秒到毫秒的时间范围内的变化。FixL谱表明，结构的动态变化分为三个阶段。配基依赖的结构动力学在最早的步骤就被观察到了。在比较这些结构变化的基础上，提出了FixL的信号转导方案。对于传感器结构域和全长蛋白质也观察到了类似的光谱变化，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活域的结构变化是耦合的。较少

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