Engineering the photoactive orange carotenoid protein with redox-controllable structural dynamics and photoprotective function.

Photosynthesis requires various photoprotective mechanisms for survival of organisms in high light. In cyanobacteria exposed to high light, the Orange Carotenoid Protein (OCP) is reversibly photoswitched from the orange (OCP) to the red (OCP) form, the latter binds to the antenna (phycobilisomes, PBs) and quenches its overexcitation. OCP accumulation implicates restructuring of a compact dark-adapted OCP state including detachment of the N-terminal extension (NTE) and separation of protein domains, which is reversed by interaction with the Fluorescence Recovery Protein (FRP). OCP phototransformation supposedly occurs via an intermediate characterized by an OCP-like absorption spectrum and an OCP-like protein structure, but the hierarchy of steps remains debatable. Here, we devise and analyze an OCP variant with the NTE trapped on the C-terminal domain (CTD) via an engineered disulfide bridge (OCP). NTE trapping preserves OCP photocycling within the compact protein structure but precludes functional interaction with PBs and especially FRP, which is completely restored upon reduction of the disulfide bridge. Non-interacting with the dark-adapted oxidized OCP, FRP binds reduced OCP nearly as efficiently as OCP devoid of the NTE, suggesting that the low-affinity FRP binding to OCP is realized via NTE displacement. The low efficiency of excitation energy transfer in complexes between PBs and oxidized OCP indicates that OCP binds to PBs in an orientation suboptimal for quenching PBs fluorescence. Our approach supports the presence of the OCP-like intermediate in the OCP photocycle and shows effective uncoupling of spectral changes from functional OCP photoactivation, enabling redox control of its structural dynamics and function.