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Vacuolar ATP-dependent proton pumps (V-ATPases) belong to a super-family of rotary ATPases and ATP synthases. The V1complex consumes ATP to drive rotation of a central rotor that pumps protons across membranes via the Vocomplex. Eukaryotic V-ATPases are regulated by reversible disassembly of subunit C, V1without C, and VO.ATP hydrolysis is thought to generate an unknown rotary state that initiates regulated disassembly. Dissociated V1is inhibited by subunit H that traps it in a specific rotational position. Here, we report the first single-molecule studies with high resolution of time and rotational position ofSaccharomyces cerevisiaeV1-ATPase lacking subunits H and C (V1ΔHC), which resolves previously elusive dwells and angular velocity changes. Rotation occurred in 120° power strokes separated by dwells comparable to catalytic dwells observed in other rotary ATPases. However, unique V1ΔHC rotational features included: 1) faltering power stroke rotation during the first 60°; 2) a dwell often occurring ∼45° after the catalytic dwell, which did not increase in duration at limiting MgATP; 3) a second dwell, ∼2-fold longer occurring 112° that increased in duration and occurrence at limiting MgATP; 4) limiting MgATP-dependent decreases in power stroke angular velocity where dwells were not observed. The results presented here are consistent with MgATP binding to the empty catalytic site at 112° and MgADP released at ∼45°, and provide important new insight concerning the molecular basis for the differences in rotary positions of substrate binding and product release between V-type and F-type ATPases.
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Visualizing Single V-ATPase Rotation Using Janus Nanoparticles
Understanding the function of rotary molecular motors, such as the rotary ATPases, relies on our ability to visualize the single-molecule rotation. Traditional imaging methods often involve tagging those motors with nanoparticles (NPs) and inferring their rotation from translational motion of NPs. Here, we report an approach using “two-faced” Janus NPs to directly image the rotation of single V-ATPase from Enterococcus hirae, an ATP-driven rotary ion pump. By employing a 500-nm silica/gold Janus NP, we exploit its asymmetric optical contrast – silica core with a gold cap on one hemisphere – to achieve precise imaging of the unidirectional counter-clockwise rotation of single V-ATPase motors immobilized on surfaces. Despite the added viscous load from the relatively large Janus NP probe, our approach provides accurate torque measurements of single V-ATPase. This study underscores the advantages of Janus NPs over conventional probes, establishing them as powerful tools for single-molecule analysis of rotary molecular motors.
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- Award ID(s):
- 2534273
- PAR ID:
- 10629863
- Publisher / Repository:
- ACS
- Date Published:
- Journal Name:
- Nano Letters
- Volume:
- 24
- Issue:
- 49
- ISSN:
- 1530-6984
- Page Range / eLocation ID:
- 15638 to 15644
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript
- Journal Article:
- https://doi.org/10.1021/acs.nanolett.4c04109
