LOV Domain Signaling

植物和藻类中参与蓝光传感的许多过程都是由具有LOV(光氧电压)结构域的酶调控的，LOV结构域包含一个黄素发色团(FMN)，其第一吸收带峰位于445 nm。在吸收蓝光光子后，半胱氨酸残基与黄素的C4a形成亚稳定的共价键。随后，酶的激活域被激活，蛋白质自动磷酸化，启动导致生物反应的信号链。关于这种激活机制的两种相互竞争的假说认为，要么是LOV结构域被光加合物形成后移开的LOV结构域阻断了活性部位，要么是LOV结构域的二聚化形成了一对酶的交叉磷酸化。该项目的主要目的是研究LOV结构域的构象动力学和LOV结构域之间的二聚体形成动力学，在暗适应和光适应状态下都是如此。Förster Resonance Energy Transfer(FRET)测量连接到蛋白质不同位置的两个染料分子之间随时间变化的距离。二聚体的形成速度可以通过标记和未标记LOV结构域的混合物的系综测量来获得。用不同的染料标记的LOV结构域可以通过荧光互相关测量来检测到共定位。单分子爆发测量将被用来测量单对FRET效率的分布。这将显示系统中构象异质性的程度。

Many processes involved in blue-light sensing in plants and algae are regulated by enzymes that possess LOV (light-oxygen-voltage) domains containing a flavin chromophore (FMN) with a first absorption band peak at 445 nm. Following absorption of a blue-light photon a cysteine residue forms a metastable covalent bond to C4a of the flavin. Subsequently, the kinase domain of the enzyme is activated and the protein autophosphorylates, initiating the signalling chain leading to the biological response. Two competing hypotheses for the mechanism of this activation suggest either blocking of the active site by the LOV domain which moves away after photoadduct formation, or cross-phosphorylation in a pair of enzymes that is formed by dimerization of LOV domains.The principal aim of the proposed project is the study of the conformational dynamics of LOV domains and the dynamics of dimer formation between LOV domains, both in the dark and light adapted states. Förster Resonance Energy Transfer (FRET) measures the time-dependent distance between two dye-molecules that will be attached to various positions of the protein. The rate of dimer formation can be obtained from ensemble measurements with mixtures of labelled and unlabelled LOV domains. Colocalization of LOV domains labeled with different dyes can be detected by fluorescence cross-correlation measurements. Single-molecule burst measurements will be used to measure the distribution of single-pair FRET efficiencies. This will show the extent of conformational heterogeneity in the system.