PMID:15339903
| Citation |
Motz, C, Hornung, T, Kersten, M, McLachlin, DT, Dunn, SD, Wise, JG and Vogel, PD (2004) The subunit b dimer of the FOF1-ATP synthase: interaction with F1-ATPase as deduced by site-specific spin-labeling. J. Biol. Chem. 279:49074-81 |
|---|---|
| Abstract |
We have used site-specific spin-labeling of single cysteine mutations within a water-soluble mutant of subunit b of the ATP synthase and employed electron spin resonance (ESR) spectroscopy to obtain information about the binding interactions of the b dimer with F1-ATPase. Interaction of b2 with a delta-depleted F1 (F1-delta) was also studied. The cysteine mutations used for spin-labeling were distributed throughout the cytosolic domain of the b subunit. In addition, each position between residues 101 and 114 of b was individually mutated to cysteine. All mutants were modified with a cysteine-reactive spin label. The room temperature ESR spectra of spin-labeled b2 in the presence of F1 or F1-delta when compared with the spectra of free b2 indicate a tight binding interaction between b2 and F1. The data suggest that b2 packs tightly to F1 between residues 80 and the C terminus but that there are segments of b2 within that region where packing interactions are quite loose. Two-dimensional gel electrophoresis confirmed binding of the modified b mutants to F1-ATPase as well as to F1-delta. Subsequent addition of delta to F1-delta.b2 complex resulted in changes in the ESR spectra, indicating different binding interactions of b to F1 in the presence or absence of delta. The data also suggest that the reconstitution of the ATP synthase is not ordered with respect to these subunits. Additional spectral components observed in b preparations that were spin-labeled between amino acid position 101 and 114 are indicative of either two populations of b subunits with different packing interactions or to helical bending within this region. |
| Links |
PubMed Online version:10.1074/jbc.M404543200 |
| Keywords |
Adenosine Triphosphatases/chemistry; Cysteine/chemistry; Cysteine/genetics; Dimerization; Electron Spin Resonance Spectroscopy; Electrophoresis, Gel, Two-Dimensional; Escherichia coli/enzymology; Free Radicals; Hydrolysis; Mutation; Protein Binding; Protein Structure, Tertiary; Proton-Translocating ATPases/chemistry; Proton-Translocating ATPases/genetics; Spin Labels; Temperature |
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