Hunke, C, Tadwal, VS, Manimekalai, MS, Roessle, M and Grüber, G (2010) The effect of NBD-Cl in nucleotide-binding of the major subunit alpha and B of the motor proteins F1FO ATP synthase and A1AO ATP synthase. J. Bioenerg. Biomembr. 42:1-10
Subunit alpha of the Escherichia coli F(1)F(O) ATP synthase has been produced, and its low-resolution structure has been determined. The monodispersity of alpha allowed the studies of nucleotide-binding and inhibitory effect of 4-Chloro-7-nitrobenzofurazan (NBD-Cl) to ATP/ADP-binding. Binding constants (K ( d )) of 1.6 microM of bound MgATP-ATTO-647N and 2.9 microM of MgADP-ATTO-647N have been determined from fluorescence correlation spectroscopy data. A concentration of 51 microM and 55 microM of NBD-Cl dropped the MgATP-ATTO-647N and MgADP-ATTO-647N binding capacity to 50% (IC(50)), respectively. In contrast, no effect was observed in the presence of N,N'-dicyclohexylcarbodiimide. As subunit alpha is the homologue of subunit B of the A(1)A(O) ATP synthase, the interaction of NBD-Cl with B of the A-ATP synthase from Methanosarcina mazei Gö1 has also been shown. The data reveal a reduction of nucleotide-binding of B due to NBD-Cl, resulting in IC(50) values of 41 microM and 42 microM for MgATP-ATTO-647N and MgADP-ATTO-647N, respectively.
4-Chloro-7-nitrobenzofurazan/pharmacology; Adenosine Diphosphate/metabolism; Adenosine Triphosphate/metabolism; Archaeal Proteins/antagonists & inhibitors; Archaeal Proteins/chemistry; Archaeal Proteins/metabolism; Bacterial Proton-Translocating ATPases/antagonists & inhibitors; Bacterial Proton-Translocating ATPases/chemistry; Bacterial Proton-Translocating ATPases/genetics; Bacterial Proton-Translocating ATPases/metabolism; Base Sequence; DNA Primers/genetics; Enzyme Inhibitors/pharmacology; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli Proteins/antagonists & inhibitors; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Kinetics; Models, Molecular; Molecular Motor Proteins/antagonists & inhibitors; Molecular Motor Proteins/chemistry; Molecular Motor Proteins/metabolism; Protein Subunits; Proton-Translocating ATPases/antagonists & inhibitors; Proton-Translocating ATPases/chemistry; Proton-Translocating ATPases/metabolism; Scattering, Small Angle; Spectrometry, Fluorescence; X-Ray Diffraction
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