Resolving stepping rotation in Thermus thermophilus H(+)-ATPase/synthase with an essentially drag-free probe

Nat Commun. 2011:2:233. doi: 10.1038/ncomms1215.

Abstract

Vacuole-type ATPases (V(o)V₁) and F(o)F₁ ATP synthases couple ATP hydrolysis/synthesis in the soluble V(1) or F₁ portion with proton (or Na(+)) flow in the membrane-embedded V(o) or F(o) portion through rotation of one common shaft. Here we show at submillisecond resolutions the ATP-driven rotation of isolated V₁ and the whole V(o)V₁ from Thermus thermophilus, by attaching a 40-nm gold bead for which viscous drag is almost negligible. V₁ made 120° steps, commensurate with the presence of three catalytic sites. Dwells between the steps involved at least two events other than ATP binding, one likely to be ATP hydrolysis. V(o)V₁ exhibited 12 dwell positions per revolution, consistent with the 12-fold symmetry of the V(o) rotor in T. thermophilus. Unlike F₁ that undergoes 80°-40° substepping, chemo-mechanical checkpoints in isolated V₁ are all at the ATP-waiting position, and V(o) adds further bumps through stator-rotor interactions outside and remote from V₁.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / biosynthesis*
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Catalytic Domain
  • Escherichia coli
  • Gold / analysis
  • Gold / metabolism
  • Hydrolysis
  • Kinetics
  • Microspheres
  • Models, Molecular
  • Molecular Probes / analysis
  • Molecular Probes / metabolism
  • Protein Binding
  • Protein Structure, Tertiary
  • Protein Subunits / chemistry
  • Protein Subunits / genetics
  • Protein Subunits / metabolism*
  • Protons
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism*
  • Rotation
  • Sodium / metabolism
  • Streptavidin / analysis
  • Streptavidin / metabolism
  • Thermus thermophilus / chemistry
  • Thermus thermophilus / enzymology
  • Thermus thermophilus / genetics
  • Vacuolar Proton-Translocating ATPases / chemistry
  • Vacuolar Proton-Translocating ATPases / genetics
  • Vacuolar Proton-Translocating ATPases / metabolism*
  • Viscosity

Substances

  • Bacterial Proteins
  • Molecular Probes
  • Protein Subunits
  • Protons
  • Recombinant Proteins
  • Gold
  • Adenosine Triphosphate
  • Streptavidin
  • Sodium
  • Vacuolar Proton-Translocating ATPases