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The Ca2+-binding photoprotein aequorin is a complex of apoAequorin (apoprotein) and (S)-2-peroxycoelenterazine. Aequorin can be regenerated by the incubation of apoAequorin with coelenterazine and molecular oxygen (O2). In this study, to investigate the molecular recognition of apoAequorin for coelenterazine using chemical probes, the chiral deaza-analogs of (S)- and (R)-deaza-CTZ (daCTZ) for coelenterazine and of (S)-2- and (R)-2-hydroxymethyl-deaza-CTZ (HM-daCTZ) for 2-peroxycoelenterazine were efficiently prepared by the improvement method. The chiral deaza-analogs of (S)-daCTZ and (S)-HM-daCTZ selectively inhibited the regeneration step to aequorin by binding the catalytic site of coelenterazine in the apoAequorin molecule. The crystal structures of the apoAequorin complexes with (S)-daCTZ and (S)-HM-daCTZ were determined, suggesting that the hydroxy moiety at the C6-hydroxyphenyl group and the carbonyl moiety of the imidazopyrazinone ring in coelenterazine are essential to bind the apoAequorin molecule through hydrogen bonding. Therefore, the chiral deaza-analogs of coelenterazine can be used as a probe to study the interaction between coelenterazine and the related proteins including photoprotein, luciferase, and coelenterazine-binding protein. [ABSTRACT FROM AUTHOR]

Vacuolar ATPases (V-ATPases) function as proton pumps in various cellular membrane systems. The hydrophilic V1 portion of the V-ATPase is a rotary motor, in which a central-axis DF complex rotates inside a hexagonally arranged catalytic A3B3 complex by using ATP hydrolysis energy. We have previously reported crystal structures of Enterococcus hirae V-ATPase A3B3 and A3B3DF (V1) complexes; the result suggested that the DF axis induces structural changes in the A3B3 complex through extensive protein-protein interactions. In this study, we mutated 10 residues at the interface between A3B3 and DF complexes and examined the ATPase activities of the mutated V1 complexes as well as the binding affinities between the mutated A3B3 and DF complexes. Surprisingly, several V1 mutants showed higher initial ATPase activities than wild-type V1-ATPase, whereas these mutated A3B3 and DF complexes showed decreased binding affinities for each other. However, the high ATP hydrolysis activities of the mutants decreased faster over time than the activity of the wild-type V1 complex, suggesting that the mutants were unstable in the reaction because the mutant A3B3 and DF complexes bound each other more weakly. These findings suggest that strong interaction between the DF complex and A3B3 complex lowers ATPase activity, but also that the tight binding is responsible for the stable ATPase activity of the complex. [ABSTRACT FROM AUTHOR]