The effect of NBD-Cl in nucleotide-binding of the major subunit α and B of the motor proteins F1FO ATP synthase and A1AO ATP synthase
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Subunit α of the Escherichia coli F1FO ATP synthase has been produced, and its low-resolution structure has been determined. The monodispersity of α 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 μM of bound MgATP-ATTO-647N and 2.9 μM of MgADP-ATTO-647N have been determined from fluorescence correlation spectroscopy data. A concentration of 51 μM and 55 μM of NBD-Cl dropped the MgATP-ATTO-647N and MgADP-ATTO-647N binding capacity to 50% (IC50), respectively. In contrast, no effect was observed in the presence of N,N′-dicyclohexylcarbodiimide. As subunit α is the homologue of subunit B of the A1AO 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 IC50 values of 41 μM and 42 μM for MgATP-ATTO-647N and MgADP-ATTO-647N, respectively.
KeywordsF1FO ATP synthase A1AO ATP synthase Subunit α Subunit B 4-Chloro-7-nitrobenzofurazan (NBD-Cl) N,N′-dicyclohexylcarbodiimide (DCCD) Small angle X-ray scattering (SAXS) Fluorescence correlation spectroscopy (FCS)
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We thank Dr. A. Balakrishna and A. Kumar for crystallographic data collection and isolation of subunit B from M. mazei Gö1, respectively. This research and the fellowship for Vikeramjeet S. Tadwal were supported by a grant from the Ministry of Education, Singapore (ARC 6/06 and RG144/06).
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