Molecular characterization of phototropin signalosome regulating photophysiological processes in green algae

Phototropin is a blue light-sensing photoreceptor, comprises two LOV domains that non-covalently bind FMN as chromophore and a serine-threonine kinase domain at its C-terminus. Phototropin is well established in higher plants and aquatic algae. However, the photoreceptor biology of the evolutionary important alga has not yet been established. We characterized the phototropin LOV domain from evolutionary important algae and compared the changes with the aquatic green algae, Chlamydomonas reinhardtii phototropin LOV domain. The intraflagellar transport (IFT) machinery responsible for bidirectional movement of proteins inwithin cilia or flagella is involved in trafficking of Chlamydomonas phototropin (CrPhot). Through immunoblotting and immunolocalization experiments with a variety of IFT mutants, we investigated the molecular details of phototropin trafficking. The CrPhot knock-out strains showed reduced photomotility, decrease level of other predicted interacting proteins like ChRs and, 14-3-3 as compared to that of the wild type strain. Various approaches like protein profiling, expression profile (qRT-PCR), and proteomics were applied to delineate its role in light-mediated physiology in C. reinhardtii. We studied the photochemistry, oligomerization, and photodynamic behaviour of LOV1 domain of a novel terrestrial algal phototropin using UV-visible, fluorescence, CD, atomic force microscopy, and time-resolved fluorescence spectroscopy techniques. Our findings highlight the unique characteristics of LOV domain in terms of very long dark recovery time. We identified the key residues responsible for this recovery kinetics in novel LOV1 protein. Different variants of LOV1 protein were produced and photochemistry was compared with the respect to the wild type protein. We proposed optogenetic potential of the engineered LOV domain in modulation of diverse cellular signalling pathways such as ciliogenesis, cell cycle, and apoptosis.