Visualizing Single V-ATPase Rotation Using Janus Nanoparticles

Abstract

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.

Keywords: Janus nanoparticles; Rotary ATPases; molecular motors; rotational tracking; single-molecule analysis.

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 biotin-streptavidin conjugation system. Further details are provided in MATERIALS AND METHODS. The black and grey dotted vertical lines indicate the rotation center and the centroid of Janus NP, respectively. The symbols $r$ and $R$ denote the rotation radius and the radius of 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) non-specifically 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. Observation was conducted at 25°C in the presence of 5 mM ATP and 300 mM NaCl. Images were recorded at 1,000 frames per second (1 ms time resolution) and are shown at 20 ms intervals. Scale bars: 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. The symbol $r$ denotes the rotation radius (the distance between the rotation center and the centroid position). (b) Distribution of the rotation radius, averaged separately for 9 molecules. (c) Time course of EhV_oV₁ rotation, with the trajectories of individual 9 molecules shown in different colors. The red-colored trajectory corresponds to the molecule shown in (a). (d) Distribution of the rotation rate for individual 9 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 $\left(P\right(\mathrm{\Delta }\theta \left)\right)$ of $\mathrm{\Delta }\theta (=\theta (t+\mathrm{\Delta }t)-\theta (t\left)\right)$ for a single EhV_oV₁ rotation probed by the Janus NP. Data were analyzed at various time intervals $\left(\mathrm{\Delta }t\right)$, each represented by a different color as indicated in the figure. (b) Plot of $\mathrm{l}\mathrm{n}\left[P\right(\mathrm{\Delta }\theta )/P(-\mathrm{\Delta }\theta \left)\right]$ as a function of $\mathrm{\Delta }\theta /k_{\mathrm{B}}T$, with colors corresponding to the time intervals in (a). (c) Torque estimated from the slopes in (b) plotted against $\mathrm{\Delta }t$. Results from three representative molecules are shown, with colors matching the time intervals in (a). (d) Distribution of torque at $\mathrm{\Delta }t=10\mathrm{m}\mathrm{s}$ (N = 9 molecules).