Molecular mechanism of light-sensor proteins containing a flavin

趋光蛋白是植物中的一种蓝光感应蛋白，LOV结构域结合黄素单核苷酸（FMN）作为发色团。光中间体状态S390是由FMN和附近的半胱氨酸之间的光诱导加合物形成的，它触发了趋光蛋白激酶激活的蛋白质结构变化。在本项目中，我们旨在揭示LOV结构域光信号转导的分子机制。通过低温FTIR光谱分析，我们发现活性半胱氨酸在Adiantum phytochrome3 （phy3-LOV2）的LOV2结构域的三激发态和非光解状态下被质子化。它的氢键相互作用在三重激发态被加强，可能与FMN发色团，这种强相互作用驱动加合物形成在微秒的时间尺度。趋光蛋白有两个LOV结构域，称为LOV1和LOV2。为什么它有两个域？一项转基因研究表明，趋光蛋白的功能只需要LOV2，而LOV1和LOV2结构域之间的x射线结构惊人地相似。我们通过紫外可见光谱和红外光谱比较了phy3的LOV1和LOV2结构域的蛋白结构变化。我们发现在LOV2中蛋白质结构的变化要比在LOV1中大得多，这与它们的功能作用是一致的。我们认为植物利用独特的蛋白质结构域（LOV结构域）调控其蛋白质结构变化来实现不同的功能。关于蛋白质结构的变化，我们之前观察到phy3-LOV2肽主链酰胺i振动区的温度依赖性FTIR光谱变化，表明蛋白质片段的结构发生了进行性变化。由于FMN还具有两个C=O基团，我们在本文中使用^<13>C标记来分配FMN和蛋白质的C=O拉伸振动。因此，通过分离发色团带，可以明确地分配温度相关的酰胺- i带。

Phototropin is a blue-light sensor protein in plants, and LOV domain binds a flavin mononucleotide (FMN) as a chromophore. A photointermediate state, S390, is formed by light-induced adduct formation between FMN and a nearby cysteine, which triggers protein structural changes for kinase activation in phototropin. In this project, we aimed at revealing the molecular mechanism of light-signal transduction in LOV domains.By means of low-temperature FTIR spectroscopy, we revealed that the reactive cysteine is protonated in the triplet-excited state of the LOV2 domain of Adiantum phytochrome3 (phy3-LOV2) as well as its unphotolyzed state. Its hydrogen-bonding interaction is strengthened in the triplet-excited state, presumably with the FMN chromophore, and such strong interaction drives adduct formation in a microsecond timescale.Phototropin has two LOV domains called LOV1 and LOV2. Why does it have two domains? A transgenic study suggested that only LOV2 is necessary in the function of phototropin, whereas X-ray structures are surprisingly similar between LOV1 and LOV2 domains. We compared protein structural changes between the LOV1 and LOV2 domains of phy3 by means of UV-visible and FTIR spevctroscopy. We found that protein structural changes are much larger in LOV2 than in LOV1, which is consistent with their functional roles. We concluded that plants utilize a unique protein architecture (LOV domain) for different functions by regulating their protein structural changes. Regarding the protein structural changes, we previously observed temperature-dependent FTIR spectral changes in the amide-I vibrational region of peptide backbone for phy3-LOV2, suggesting the progressive structural changes in the protein moiety. Since FMN also possesses two C=O groups, we assigned C=O stretching vibrations of FMN and protein by using ^<13>C-labeling in this article. Consequently, temperature-dependent amide-I bands are unequivocally assigned by separating the chromophore bands.