Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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4-Chloro-7-nitrobenzofurazan / pharmacology*
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Adenosine Diphosphate / metabolism
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Adenosine Triphosphate / metabolism
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Archaeal Proteins / antagonists & inhibitors
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Archaeal Proteins / chemistry*
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Archaeal Proteins / metabolism*
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Bacterial Proton-Translocating ATPases / antagonists & inhibitors
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Bacterial Proton-Translocating ATPases / chemistry*
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Bacterial Proton-Translocating ATPases / genetics
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Bacterial Proton-Translocating ATPases / metabolism*
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Base Sequence
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DNA Primers / genetics
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Enzyme Inhibitors / pharmacology
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Escherichia coli / enzymology
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Escherichia coli / genetics
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Escherichia coli Proteins / antagonists & inhibitors
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Escherichia coli Proteins / chemistry*
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Escherichia coli Proteins / genetics
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Escherichia coli Proteins / metabolism*
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Kinetics
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Models, Molecular
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Molecular Motor Proteins / antagonists & inhibitors
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Molecular Motor Proteins / chemistry
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Molecular Motor Proteins / metabolism
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Protein Subunits
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Proton-Translocating ATPases / antagonists & inhibitors
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Proton-Translocating ATPases / chemistry*
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Proton-Translocating ATPases / metabolism*
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Scattering, Small Angle
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Spectrometry, Fluorescence
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X-Ray Diffraction
Substances
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Archaeal Proteins
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DNA Primers
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Enzyme Inhibitors
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Escherichia coli Proteins
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Molecular Motor Proteins
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Protein Subunits
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Adenosine Diphosphate
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Adenosine Triphosphate
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A1A0 ATPase, Methanosarcina mazei
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Bacterial Proton-Translocating ATPases
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Proton-Translocating ATPases
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atpB protein, E coli
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4-Chloro-7-nitrobenzofurazan