The subunit b dimer of the FOF1-ATP synthase: interaction with F1-ATPase as deduced by site-specific spin-labeling.
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.
Motz
Christian
C
Department of Biological Sciences, Southern Methodist University, Dallas Texas 75275, USA.
Hornung
Tassilo
T
Kersten
Michael
M
McLachlin
Derek T
DT
Dunn
Stanley D
SD
Wise
John G
JG
Vogel
Pia D
PD
D000251:Adenosine Triphosphatases / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D019281:Dimerization; D004578:Electron Spin Resonance Spectroscopy; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000201:enzymology*; D005609:Free Radicals; D006868:Hydrolysis; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics*; D013113:Spin Labels; D013696:Temperature
D005609:Free Radicals; D013113:Spin Labels; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases; D003545:Cysteine