LOV Domain Signaling

在植物和藻类中，许多与蓝光感应有关的过程是由具有LOV（光氧电压）结构域的酶调控的，该结构域含有黄素发色团（FMN），其第一吸收带峰位于445 nm。吸收蓝光光子后，半胱氨酸残基与黄素的C4a形成亚稳共价键。随后，该酶的激酶结构域被激活，蛋白质自磷酸化，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设认为，在光加合物形成后，LOV结构域会阻断活性位点，或者在LOV结构域二聚化形成的一对酶中发生交叉磷酸化。该项目的主要目的是研究在黑暗和光明适应状态下LOV结构域的构象动力学和LOV结构域之间二聚体形成的动力学。Förster共振能量转移（FRET）测量两个染料分子之间的时间依赖距离，这些染料分子将附着在蛋白质的不同位置。二聚体的形成速率可以通过标记和未标记的LOV结构域的混合物的系综测量得到。用不同染料标记的LOV结构域的共定位可以通过荧光互相关测量来检测。单分子爆发测量将用于测量单对FRET效率的分布。这将显示系统中构象非均质性的程度。

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.