Visualizing Single V-ATPase Rotation Using Janus Nanoparticles

Understanding the function of rotary molecular motors, such as rotary ATPases, relies on our ability to visualize single-molecule rotation. Traditional imaging methods often involve tagging those motors with nanoparticles (NPs) and inferring their rotation from the translational motion of NPs. Here, we report an approach using “two-faced” Janus NPs to directly image the rotation of a 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, a silica core with a gold cap on one hemisphere, to achieve precise imaging of the unidirectional counterclockwise 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 a single V-ATPase. This study underscores the advantages of Janus NPs over conventional probes, establishing them as powerful tools for the single-molecule analysis of rotary molecular motors.