- Reactive oxygen species are key factors that strongly affect the cellular redox state and regulate various physiological and cellular phenomena. To monitor changes in the redox state, we previously developed fluorescent redox sensors named Re-Q, the emissions of which are quenched under reduced conditions. However, such fluorescent probes are unsuitable for use in the cells of photosynthetic organisms because they require photoexcitation that may change intracellular conditions and induce autofluorescence, primarily in chlorophylls.
- The FoF1 synthase produces ATP from ADP and inorganic phosphate. The γ subunit of FoF1 ATP synthase in photosynthetic organisms, which is the rotor subunit of this enzyme, contains a characteristic β-hairpin structure. This structure is formed from an insertion sequence that has been conserved only in phototrophs. Using recombinant subcomplexes, we previously demonstrated that this region plays an essential role in the regulation of ATP hydrolysis activity, thereby functioning in controlling intracellular ATP levels in response to changes in the light environment.
- pH is one of the most critical physiological parameters determining vital cellular activities, such as photosynthetic performance. Fluorescent sensor proteins capable of measuring in situ pH in animal cells have been reported. However, these proteins require an excitation laser for pH measurement that may affect photosynthetic performance and induce autofluorescence from chlorophyll. As a result, it is not possible to measure the intracellular or intraorganelle pH changes in plants. To overcome this problem, we developed a luminescent pH sensor by fusing the luminescent protein Nanoluc to a uniquely designed pH-sensitive GFP variant protein.
- ATP hydrolysis activity catalyzed by chloroplast and proteobacterial ATP synthase is inhibited by their ε subunits. To clarify the function of the ε subunit from phototrophs, here we analyzed the ε subunit–mediated inhibition (ε-inhibition) of cyanobacterial F1-ATPase, a subcomplex of ATP synthase obtained from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. We generated three C-terminal α-helix null ε-mutants; one lacked the C-terminal α-helices, and in the other two, the C-terminal conformation could be locked by a disulfide bond formed between two α-helices or an α-helix and a β-sandwich structure.