Blue-light induced interaction of LOV domains from Chlamydomonas reinhardtii

The phototropin from Chlamydomonas reinhardtii is a 120 kDa blue light receptor that plays a key role in gametogenesis of this green alga. It comprises two light-sensing domains termed LOV1 and LOV2 (light oxygen and voltage) and a serine/threonine kinase domain. The post-translationally incorporated chromophore is flavin mononucleotide (FMN). Upon absorption of blue light, LOV domains undergo a photocycle that activates a Ser/Thr kinase. The mechanism of this activation is still unknown. We studied the oligomerization of the recombinant LOV1 domain (amino acids 16-133) of C. reinhardtii by means of UV/Vis spectroscopy, size exlusion chromatography (SEC) and chemical cross linking with glutardialdehyde. The thermal back reaction of LOV1 from the signaling state to the dark state as monitored by UV/Vis spectroscopy after an intensive blue light pulse could not be explained by a monoexponential model, although the spectra did not indicate the presence of an additional species. Therefore, we investigated the quaternary structure of the LOV1 domain by size-exlusion chromatography in the dark. This revealed an equilibrium between dimers and higher oligomers (M-w > 200 kDa) under native conditions. No monomers were detected by SEC. However, by analysis of the equilibrium by cross-linking of the protein with glutaraldehyde and subsequent SDS-PAGE, monomers and dimers were identified. Exposure to LOV1 to blue light resulted in a decrease in the monomer/dimer ratio. A model is presented that accounts for a blue-light driven change in the quaternary structure of the LOV1 domain and gives hints to the molecular basis of light activation and regulation in LOV-containing proteins.