Abstract
The angular velocities of ATPase-dependent power strokes as a function of the rotational position for the A-type molecular motor A3B3DF, from the Methanosarcina mazei Gö1 A-ATP synthase, and the thermophilic motor α3β3γ, from Geobacillus stearothermophilus (formerly known as Bacillus PS3) F-ATP synthase, are resolved at 5 μs resolution for the first time. Unexpectedly, the angular velocity profile of the A-type was closely similar in the angular positions of accelerations and decelerations to the profiles of the evolutionarily distant F-type motors of thermophilic and mesophilic origins, and they differ only in the magnitude of their velocities. M. mazei A3B3DF power strokes occurred in 120° steps at saturating ATP concentrations like the F-type motors. However, because ATP-binding dwells did not interrupt the 120° steps at limiting ATP, ATP binding to A3B3DF must occur during the catalytic dwell. Elevated concentrations of ADP did not increase dwells occurring 40° after the catalytic dwell. In F-type motors, elevated ADP induces dwells 40° after the catalytic dwell and slows the overall velocity. The similarities in these power stroke profiles are consistent with a common rotational mechanism for A-type and F-type rotary motors, in which the angular velocity is limited by the rotary position at which ATP binding occurs and by the drag imposed on the axle as it rotates within the ring of stator subunits.
Highlights
The angular velocities of ATPase-dependent power strokes as a function of the rotational position for the A-type molecular motor A3B3DF, from the Methanosarcina mazei Go1 A-ATP synthase, and the thermophilic motor ␣33␥, from Geobacillus stearothermophilus F-ATP synthase, are resolved at 5 s resolution for the first time
In F-type motors, elevated ADP induces dwells 40° after the catalytic dwell and slows the overall velocity. The similarities in these power stroke profiles are consistent with a common rotational mechanism for A-type and F-type rotary motors, in which the angular velocity is limited by the rotary position at which ATP binding occurs and by the drag imposed on the axle as it rotates within the ring of stator subunits
Removal of all cysteines in the complex except for A71C via amino acid substitutions C36V, C72V, C181A, and C380V in subunit A as well as C67S in subunit B resulted in a decrease in ATPase activity of 54%. Because these cysteines contribute significantly to the hydrolytic activity of the enzyme, and are not accessible for attachment of the neutravidin-coated gold nanorod, the His8tagged A3B3DA71CF complex of the M. mazei Go1 A-ATP synthase was used for single molecule rotation experiments
Summary
ATPase Activity of the A3B3DF Complex from the Archaeal M. mazei Goand Its Mutants—The A-ATP synthase complexes of wild type MmA3B3DF and its mutants MmA3B3DF-M1 and MmA3B3DF-M2 were purified as described recently [25]. Removal of all cysteines in the complex except for A71C via amino acid substitutions C36V, C72V, C181A, and C380V in subunit A as well as C67S in subunit B (mutant MmA3B3DF-M1) resulted in a decrease in ATPase activity of 54% Because these cysteines contribute significantly to the hydrolytic activity of the enzyme, and are not accessible for attachment of the neutravidin-coated gold nanorod, the His8tagged A3B3DA71CF complex of the M. mazei Go1 A-ATP synthase was used for single molecule rotation experiments. This complex will be subsequently referred to as the MmA3B3DF complex. Rotation of the nanorod was observed as sinusoidal changes in red light intensity [26, 27]
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