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.
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.
Hunke
Cornelia
C
School of Biological Sciences, Nanyang Technological University, Singapore, 637551, Republic of Singapore.
Tadwal
Vikeramjeet Singh
VS
Manimekalai
Malathy Sony Subramanian
MS
Roessle
Manfred
M
Grüber
Gerhard
G
D009327:4-Chloro-7-nitrobenzofurazan / Q000494:pharmacology*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D019843:Archaeal Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D025243:Bacterial Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D020409:Molecular Motor Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D021122:Protein Subunits; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D053838:Scattering, Small Angle; D013050:Spectrometry, Fluorescence; D014961:X-Ray Diffraction
D019843:Archaeal Proteins; D017931:DNA Primers; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D020409:Molecular Motor Proteins; D021122:Protein Subunits; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; C113203:A1A0 ATPase, Methanosarcina mazei; D025243:Bacterial Proton-Translocating ATPases; D006180:Proton-Translocating ATPases; C074428:atpB protein, E coli; D009327:4-Chloro-7-nitrobenzofurazan