2023

Kurttila, Moona

Phytochromes are red and far-red light sensing photoreceptors found in plants, bacteria, algae and fungi. They translate light cues into biochemical signalling cascades and thus allow organisms to adapt to the environment. They control growth and reproduction of nearly all vegetation, and have enormous potential for optogenetic applications to control desired cellular events by light. Light absorption causes isomerization of a bilin chromophore, which triggers structural rearrangements of the protein, ultimately controlling the biochemical activity of the system. In this thesis, spectroscopic, structural and biochemical data were combined to understand the function of a bacterial phytochrome from Deinococcus radiodurans. The system consists of a conserved photosensory module (PSM), which has a chromophore binding domain (CBD) and a phytochrome specific (PHY) domain. The PHY domain structurally and functionally connects the chormophore in CBD to the biologically active output module (OPM). Moreover, it includes a structurally flexible hairpin extension, "tongue", that extends to the vicinity of the chromophore and refolds during photoconversion. In the experimental part of the work, a method suitable to study dynamics of the tongue with UV–vis spectroscopy was established. With this method, combined with activity assays and other spectroscopic approaches, the dynamics of the tongue were shown to be dictated by the coupling with the CBD and stabilized in a complete system by the OPM. The tongue dynamics do not virtually affect the chromophore environment in the dark state, but were shown to be necessary for the thermal stability of the full-length system. The tongue is also crucial for controlling the biochemical activity of the system by light. Spectroscopic measurements suggested that in a tongueless systems, light signal becomes trapped within the CBD, which prohibits (de)activation of the OPM. During the photocycle, the tongue was shown to be in action in all intermediate states using site-selective labeling and timeresolved vibrational spectroscopy. The profound characterization of the tongue properties and dynamics with biophysical as well as biochemical approaches in this thesis revealed that the tongue is a key component in phytochrome allostery.