Single molecule studies of F₁-ATPase with a truncated gamma subunit

制度:新 ; 文部省報告番号:乙2156号 ; 学位の種類:博士(理学) ; 授与年月日:2008/2/25 ; 早大学位記番号:新472

Neither Helix in the Coiled Coil Region of the Axle of F1-ATPase Plays a Significant Role in Torque Production

F1-ATPase is an ATP-driven rotary molecular motor in which the central γ-subunit rotates inside the cylinder made of α3β3 subunits. The amino and carboxy termini of the γ-subunit form the axle, an α-helical coiled coil that deeply penetrates the stator cylinder. We previously truncated the axle step by step, starting with the longer carboxy terminus and then cutting both termini at the same levels, resulting in a slower yet considerably powerful rotation. Here we examine the role of each helix by truncating only the carboxy terminus by 25–40 amino-acid residues. Longer truncation impaired the stability of the motor complex severely: 40 deletions failed to yield rotating the complex. Up to 36 deletions, however, the mutants produced an apparent torque at nearly half of the wild-type torque, independent of truncation length. Time-averaged rotary speeds were low because of load-dependent stumbling at 120° intervals, even with saturating ATP. Comparison with our previous work indicates that half the normal torque is produced at the orifice of the stator. The very tip of the carboxy terminus adds the other half, whereas neither helix in the middle of the axle contributes much to torque generation and the rapid progress of catalysis. None of the residues of the entire axle played a specific decisive role in rotation

The Rotor Tip Inside a Bearing of a Thermophilic F(1)-ATPase Is Dispensable for Torque Generation

F(1)-ATPase is an ATP-driven rotary molecular motor in which the central γ-subunit rotates inside a stator cylinder made of α(3)β(3) subunits. To elucidate the role of rotor-stator interactions in torque generation, we truncated the γ-subunit at its carboxyl terminus, which forms an α helix that penetrates deeply into the stator cylinder. We used an α(3)β(3)γ subcomplex of F(1)-ATPase derived from thermophilic Bacillus PS3 and expressed it in Escherichia coli. We could obtain purified subcomplexes in which 14, 17, or 21 amino-acid residues were deleted. The rotary characteristics of the truncated mutants, monitored by attaching a duplex of 0.49-μm beads to the γ-subunit, did not differ greatly from those of the wild-type over the ATP concentrations of 20 nM–2 mM, the most conspicuous effect being ∼50% reduction in torque and ∼70% reduction in the rate of ATP binding upon deletion of 21 residues. The ATP hydrolysis activity estimated in bulk samples was more seriously affected. The 21-deletion mutant, in particular, was >10-fold less active, but this is likely due to instability of this subcomplex. For torque generation, though not for rapid catalysis, most of the rotor-stator contacts on the deeper half of the penetrating portion of the γ-subunit are dispensable

Torque Generation in F1-ATPase Devoid of the Entire Amino-Terminal Helix of the Rotor That Fills Half of the Stator Orifice

F1-ATPase is an ATP-driven rotary molecular motor in which the central γ-subunit rotates inside a cylinder made of α3β3 subunits. The amino and carboxyl termini of the γ rotor form a coiled coil of α-helices that penetrates the stator cylinder to serve as an axle. Crystal structures indicate that the axle is supported by the stator at two positions, at the orifice and by the hydrophobic sleeve surrounding the axle tip. The sleeve contacts are almost exclusively to the longer carboxyl-terminal helix, whereas nearly half the orifice contacts are to the amino-terminal helix. Here, we truncated the amino-terminal helix stepwise up to 50 residues, removing one half of the axle all the way up and far beyond the orifice. The half-sliced axle still rotated with an unloaded speed a quarter of the wild-type speed, with torque nearly half the wild-type torque. The truncations were made in a construct where the rotor tip was connected to a β-subunit via a short peptide linker. Linking alone did not change the rotational characteristics significantly. These and previous results show that nearly half the normal torque is generated if rotor-stator interactions either at the orifice or at the sleeve are preserved, suggesting that the make of the motor is quite robust