Visualizing Single V-ATPase Rotation Using Janus Nanoparticles

Abstract

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.

Keywords: Janus nanoparticles; fluctuation theorem; rotary ATPases; rotational tracking; single-molecule analysis; torque measurement.

Figure 1

(a) Schematic of the Janus NP preparation process. (b) Schematic illustration of single-molecule imaging of EhV_oV₁ rotation using a Janus NP probe. The rotor c-ring of EhV_oV₁ is immobilized on the Ni-NTA-coated coverslip via His₃ tags, while the single Janus NP is attached to the stator A subunit of EhV_oV₁ via the biotin–streptavidin conjugation system. Further details are provided in Materials and Methods. The black and gray dotted vertical lines indicate the rotation center and the centroid of the Janus NP, respectively. r and R denote the rotation radius and the radius of the Janus NP, respectively.

Figure 2

Imaging orientation of aminated silica and Janus NPs. (a) Phase-contrast images of a single aminated silica NP (top) and a single Janus NP (bottom) nonspecifically attached to the glass coverslip. (b) Time-lapse phase-contrast images demonstrating the rotational motion of a single Janus NP specifically attached to EhV_oV₁, driven by ATP hydrolysis. The observation was conducted at 25 °C in the presence of 5 mM ATP and 300 mM NaCl. Images were recorded at a rate of 1000 frames per second (1 ms time resolution) and are shown at 20 ms intervals. The scale bars are 500 nm.

Figure 3

Single-molecule analysis of EhV_oV₁ rotation probed by Janus NPs. (a) Example of the centroid position distribution of the bright contrast region of the Janus NP. r denotes the rotation radius (the distance between the rotation center and the centroid position). (b) Distribution of the rotation radius, averaged separately for nine molecules. (c) Time course of EhV_oV₁ rotation, with the trajectories of nine individual molecules shown in different colors. The red-colored trajectory corresponds to the molecule shown in panel a. (d) Distribution of the rotation rate for nine individual molecules. The rotation was observed in the presence of 5 mM ATP and 300 mM NaCl, with experiments conducted in three independent trials.

Figure 4

Estimation of torque using the fluctuation theorem. (a) Probability distribution [P(Δθ)] of Δθ [=θ(t + Δt) – θ(t)] for a single EhV_oV₁ rotation probed by the Janus NP. Data were analyzed at various time intervals (Δt), each represented by a different color as indicated in the figure. (b) Plot of ln[P(Δθ)/P(−Δθ)] as a function of Δθ/k_BT, with colors corresponding to the time intervals in panel a. (c) Torque estimated from the slopes in panel b plotted vs Δt. Results from three representative molecules are shown, with colors matching the time intervals in panel a. (d) Distribution of torque with a Δt of 10 ms (N = 9 molecules).