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.
