Lukashev, E. P. | bioRxiv Science

bioRxiv2018

Functional interaction of low-homology FRPs from different cyanobacteria with Synechocystis OCP

Photosynthesis requires a balance between efficient light harvesting and protection against photodamage. The cyanobacterial photoprotection system uniquely relies on the functioning of the photoactive orange carotenoid protein (OCP) that under intense illumination provides fluorescence quenching of the light-harvesting antenna complexes, phycobilisomes. The recently identified fluorescence recovery protein (FRP) binds to the photoactivated OCP and accelerates its relaxation into the basal form, completing the regulatory circle. The molecular mechanism of FRP functioning is largely controversial. Moreover, since the available knowledge has mainly been gained from studying Synechocystis proteins, the cross-species conservation of the FRP mechanism remains unexplored. Besides phylogenetic analysis, we performed a detailed structural-functional analysis of two selected low-homology FRPs by comparing them with Synechocystis FRP (SynFRP). While adopting similar dimeric conformations in solution and preserving binding preferences of SynFRP toward various OCP variants, the low-homology FRPs demonstrated distinct binding stoichiometries and differentially accentuated features of this functional interaction. By providing clues to understand the FRP mechanism universally, our results also establish foundations for upcoming structural investigations necessary to elucidate the FRP-dependent regulatory mechanism.

biochemistry↗

bioRxiv2017

The photocycle of orange carotenoid protein conceals distinct intermediates and asynchronous changes in the carotenoid and protein components

The 35 kDa water-soluble Orange Carotenoid Protein (OCP) is responsible for photoprotection in cyanobacteria. It acts as a light intensity sensor that simultaneously serves as efficient quencher of phycobilisome excitation energy as well as of reactive oxygen species. Photoactivation triggers large-scale conformational rearrangements to convert OCP from the orange OCPO state to the red active signaling state OCPR, as demonstrated by various structural methods. Eventually, such rearrangements imply complete yet reversible separation of structural domains (C- and N-terminal domain) and significant translocation of the carotenoid cofactor. Very recently, dynamic crystallography of OCPO crystals suggested the existence of photocycle intermediates with small-scale rearrangements that may trigger further transitions in the protein. However, the currently existing gap between the ultra-fast picosecond and 100 millisecond time scale of spectroscopic and structural data precludes knowledge about distinct intermediate states. In this study, we took advantage of single 7 ns laser pulses to study carotenoid absorption transients in OCP on the time-scale from 100 ns to 10 s, which allowed us to detect a red intermediate state preceding the red signaling state OCPR. In addition, time-resolved fluorescence spectroscopy and following assignment of carotenoid-induced quenching of different tryptophan residues revealed a novel orange intermediate state, which appears during back-relaxation of photoactivated OCPR to OCPO. Our results show asynchronous changes in the carotenoid and protein components and provide refined mechanistic information about the OCP photocycle as well as introduce new kinetic signatures for future studies of OCP photoactivity and photoprotection.\n\nSignificance statementCyanobacteria utilize the Orange Carotenoid Protein (OCP) to protect their photosynthetic apparatus from the harmful effects of intense sunlight. OCP is a blue light-triggered photoswitch, which undergoes photoconversion from its dark adapted orange to the active red state, the latter being able to interact with the phycobilisome antennae and quench their fluorescence, thus avoiding excessive energy flow to the photosystems. With the help of the fluorescence recovery protein (FRP), OCP detaches from phycobilisomes and can return faster into the orange state. Until now, only the thermodynamically stable orange state and the metastable red state are established in a primitive photocycle. In this work, we apply transient absorption and fluorescence spectroscopy and identify two novel photocycle intermediates of physiological relevance.

biophysics↗