To generate S. cerevisiae that express the V₁-ATPase for single molecule measurements, the regulatory subunits C and H were deleted, two cysteine substitution mutations (Y73C and T123C) were made on subunit D for biotinylation, and a 6xhis tag was added to c-terminus of subunit G for purification.

First, the genes encoding subunits C and H (VMA5 and VMA13, respectively) in the S. cerevisiae strain SF838–5Aα genomic DNA were replaced with NAT and KanMX selectable marker genes through homologous recombination, respectively. Additionally, the subunit G gene (VMA10) was replaced with the URA3 gene so the wild type subunit G would not compete for assembly in V₁ because only the mutant containing the 6xhis tag was expressed. Mutant colonies were selected by inoculating the cells on SC + nourseothricin + kanamycin-uracil plates.

To generate V₁ with a 6xhis tag on subunit G, yeast genomic DNA was isolated from SF838–5Aα cells. The subunit G gene (VMA10) was PCR amplified using primers that contained the His tag sequence, the PCR product was restriction digested, and the DNA fragment was subcloned the expression vector. Then, 5Aα VMA5Δ::NAT, VMA10Δ::URA3, VMA13Δ::KanMX cells were transformed with the recombinant plasmid. Mutant colonies were selected by inoculating the cells on SC-uracil-leucine plates.

To generate the cysteine substitution mutation for the biotinylation, the subunit D gene (VMA8) was PCR amplified from the genomic DNA, the PCR product was restriction digested, and the DNA fragment was subcloned into the pRS313 expression vector. The two cysteine substitution mutations (Y73C and T123C) were introduced through site-directed mutagenesis. This plasmid was used as a template to PCR amplify the mutant VMA8 gene along with the HIS3 gene from the vector using primers containing the 5′ and 3’ flanking regions of the VMA8 gene. Then, the double cysteine mutant VMA8-HIS3 PCR product was integrated into the genome of 5Aα VMA5Δ::NAT, VMA10Δ::URA3, VMA13Δ::KanMX cells containing pRS315-VMA10-6His through homologous recombination. Mutant colonies were selected by inoculating the cells on SC-uracil-leucine-histidine plates. Mutations were confirmed after each step with agarose gel electrophoresis and DNA sequencing.

Saccharomyces cerevisiae 5Aα VMA5Δ::NAT, VMA8-Y73C-T123C-HIS3, VMA10Δ::URA3, VMA13Δ::KanMX - pRS315-VMA10-6His cells were grown in six 1L SC-histidine-leucine-uracil growth media at 30°C while shaking until the OD₆₀₀ was on average 1.0 OD/mL. The cells were spun down at 5,000 rpm at room temperature for 5 min. The cell pellets were resuspended in 200 mL of spheroplast pretreatment buffer (100 mM Tris/HCl pH 9.4, 10 mM DTT) and spun down in the same condition. The cells were resuspended in 2% glucose solution and spun down. The cells were resuspended in spheroplast buffer (10 mM Tris/HCl pH 7.5, 1.2 M sorbitol, 40% glucose) to the final concentration of 15 OD/mL. Then, 1.5 U/μL zymolyase was added to the cell suspension to the final concentration of 1U/10 OD of cells. The cells were incubated at 30°C while shaking at 80 rpm for 60 min. After incubation, the spheroplasts were spun down at 3,000 rpm in 4°C for 5 min. The pellets were resuspended in spheroplast wash buffer and spun down in the same condition and the washing step was repeated two more times (spheroplast wash buffer: 6.8 mg/mL Yeast Nitrogen Base, 50 mM sodium phosphate dibasic, 50 mM succinic acid/NaOH pH 5.0, 2% glucose, 1.2 M sorbitol, 0.02 mg/mL histidine, 0.12 mg/mL leucine, 0.02 mg/mL adenine, 0.06 mg/mL lysine, 0.02 mg/mL arginine, 0.02 mg/mL tryptophan, 0.03 mg/mL tyrosine, 0.2 mg/mL threonine, 0.02 mg/mL methionine, 0.05 mg/mL phenylalanine, 0.02 mg/mL uracil). The following steps were done at 4°C. The cell pellet was homogenized in 20 mL of lysis buffer (PBS +1% (w/v) C₁₂E₉ 1mM PMSF, 5 μg/mL aprotinin, 2 μg/mL chymostatin, 1 μg/mL pepstatin A, 1 μg/mL leupeptin) and incubated in ice for 10 min. The samples were centrifuged at 30 k rpm (109,000 xg). After centrifugation, 10X binding buffer (0.5 M Tris/HCl pH 8.0, 1 M KCl, 400 mM imidazole, 50 mM MgCl₂) was added to the supernatant to make the final concentration 1X. Finally, V₁-ATPase was purified from the mixture by Ni-NTA chromatography, 1 mg of biotin maleimide was added to the column elution and incubated at 4°C for 15 min while shaking, and the sample was run through Sephadex G50 column equilibrated with storage buffer (50 mM Tris/HCl pH 8.0, 20 mM KCl, 2 mM ATP, 1 mM MgCl₂, 15% glycerol). The purified, biotinylated V₁-ATPase samples were aliquoted into 20 μL, quickly frozen, and stored at −80°C until use.

The rate of ATP hydrolysis by the purified V₁ΔHC was measured with an ATP-regenerating NADH-coupled assay (Lotscher et al., 1984). The measurement was made with the final concentration of 25 mM Tris/HCl (pH 8.0), 60 mM KCl, 2.5 mM phosphoenolpyruvate, 0.3 mM NADH, 17.5 Units pyruvate kinase (rabbit muscle, Sigma Aldrich), 25 Units L-lactate dehydrogenase (rabbit muscle, Sigma Aldrich), at varying concentrations of ATP including 1, 0.5, 0.2, 0.1, 0.05, 0.02, 0.01 mM, twice the MgCl₂ concentration for each corresponding ATP concentration, and 3.22 × 10⁻⁵ mM of purified V₁ΔHC (0.0174 mg/mL) in a final volume of 2.5 mL. The rate was determined in three replicates as the change in absorbance at 340 nm using a Cary 100 spectrophotometer with Peltier temperature control at 25°C. MgATP concentration was determined by the Maxchelator program MgATP calculator v1.3 using constants from NIST database #46 v8 (UC Davis Health).

The rotation of individual V₁ΔHC molecules were observed with a single-molecule rotation assay using gold nanorods under a dark field microscope (Spetzler et al., 2009; Martin et al., 2014). Purified V₁ΔHC molecules were immobilized on a microscope cover slip by the His-tag on the G-subunits, unbound molecules were washed off the slide with wash buffer (30 mM Tris/HCl pH 8.0, 10 mM KCl). The surface area of the cover slip that remained exposed around the bound V₁ΔHC molecules was then coated with BSA-C, which prevented the gold nanorods from binding nonspecifically to the surface. The 80 × 40 nm gold nanorod (A12-50-600 purchased from Nanopartz) coated with Neutravidin was bound to the biotinylated subunit D, excess gold nanorods were washed off with the wash buffer, and rotation buffer (1 mM MgCl₂, 2 mM ATP, 30 mM Tris/HCl pH 8.0, 10 mM KCl) was added to the cover slip (Spetzler et al., 2009). The rotations of individual molecules were observed by measuring the fluctuation of polarized red light scattered off the AuNR using a single-photon detector. In each molecule observed, the orientation of the polarizing filter was adjusted to align with the minimum light intensity position that corresponded to one of the three catalytic dwells. The sinusoidal fluctuation of the polarized red-light intensity was measured as the gold nanorod rotated from 0° to 90° relative to the catalytic dwell position. Measurements were taken in the form of 5 s datasets at a frame rate of 100 kHz. The standard error measurements of histograms of the intensity of red light scattered from a single nonrotating nanorod fixed to a slide as a function of the rotational position of the polarizer varies between about 0.02 and 0.12° as the scattered light intensity varied between minimum and maximum values (Ishmukhametov et al., 2010).

The ATPase activity of purified V₁ΔHC versus the MgATP concentration (Figure 2A) was measured using an ensemble coupled assay with pyruvate kinase and lactic dehydrogenase at 25°C. The apparent V_max was observed at 990 μM MgATP. The double-reciprocal plot of ATPase activity versus [MgATP] was not linear (Figure 2B). As such, V₁ΔHC did not exhibit simple Michaelis-Menten kinetics, which would have a linear dependence in a double reciprocal plot defined by Eq. 1, 1 / v = K M / V max 1 / MgATP + 1 / V max (1) where v is the observed velocity at a given MgATP concentration, V_max is the apparent maximum velocity and K_M is the Michaelis constant. The kinetic values were determined from Eq. 1 for each of three consecutive ATP concentrations in the double-reciprocal plot. The V_max increased with MgATP concentration to a maximum turnover number of 9.6 s⁻¹ (Figure 2C, inset). The time required to hydrolyze an ATP at 990 μM MgATP (saturating) as well as at 490 μM and 5.7 μM MgATP, which were rate-limiting, is shown in Table 1. The V_max/K_M values decreased 30-fold versus MgATP in a non-linear manner (Figure 2C), which indicates that the affinity of the empty catalytic site to bind MgATP decreases as V₁ΔHC approaches saturating MgATP concentrations. At saturating MgATP concentrations, the K_M was 41.7 μM. Changes in both V_max (∼2-fold) and K_M (100-fold) contributed to the decrease in V_max/K_M versus MgATP.

To measure ATP hydrolysis-dependent rotation of subunit D, purified V₁ΔHC molecules were immobilized on a microscope cover slip by the His-tags on the three G subunits, and a 35 × 75 nm NeutrAvidin-coated gold nanorod (AuNR) was attached to the biotinylated Y73C and T123C mutations of subunit D (Figure 3A). Rotation of single V₁ΔHC molecules was observed in the presence of MgATP by the changes in intensity of polarized red light that scatters from the AuNR. While concentrations of the MgATP complex used in ensemble ATPase measurements and single-molecule experiments were the same, the Mg:ATP ratio was 2:1 and 1:2, respectively. The 1:2 ratio used in the single-molecule experiments was intentional to minimize MgADP inhibition. These measurements were also carried out in the absence of an ATP regenerating system because the small number of V₁ molecules on the cover slip did not consume significant amounts of ATP during the assay. After the scattered red light passed through a high wavelength band-pass filter to remove light of wavelengths shorter than 600 nm, and a polarizing filter that could be rotated to specific orientations, the light intensity changes were quantified by an avalanche photodiode with a 50 ns time resolution. In this manner the intensity of scattered red light was at a minimum and maximum when the polarizer was oriented perpendicular and parallel to the long axis of the AuNR (Figure 3B).

Figure 4 shows the results of a polarizer rotation measurement when the AuNR is attached to subunit D of a single V₁ΔHC molecule. In this experiment, the light intensity scattered from the polarizer was rotated 10° in successive 5 s intervals for a total of 360°. During this time, the intensity of light scattered from the AuNR was acquired by the single-photon counter at 1 kHz (equivalent to 1,000 fps). Since red light scatters specifically from the long axis of the AuNR, the scattered light intensity when subunit D was not rotating in this experiment (Figure 4A, control) varied in a sinusoidal manner relative to the rotational axis of the polarizer (Spetzler et al., 2006; Ishmukhametov et al., 2010; Frasch et al., 2022).

In the presence of 990 μM (saturating) MgATP (Figure 4B), three sinusoidal intensity curves were observed in the polarizer rotation measurement, which were off-set from each other by 120°. The 1 kHz data acquisition rate provides a time resolution of 1 ms such that the light intensities reported the rotational position of the AuNR primarily when it was in the same position for more than 1 ms. This indicates that, in the presence of saturating MgATP, subunit D stopped at three rotary positions separated by 120° that each lasted >1 ms. To do so, subunit D rotated between these dwells with a power stroke that occurred too fast for the 1 kHz data acquisition rate to record as much scattered light as was observed during the dwells. These dwells are hereafter referred to as catalytic dwells, since they are comparable to the duration and rotary positions of catalytic dwells observed between the 120° ATPase-driven power strokes of F₁, A₁, V/A₁ and bacterial V₁ ATPases (Spetzler et al., 2006; Furuike et al., 2011; Minagawa et al., 2013; Sielaff et al., 2016; Zarco-Zavala et al., 2020).

To resolve the intermediate positions of the 120° rotational events between the dwells observed in Figure 4, the intensity of light scattered from the AuNR was sampled at 200 kHz (5 μs per data point). Prior to the 5 s data acquisition of each V₁ΔHC molecule, the rotational position of the polarizer was set so that the scattered light intensity was at a minimum at one of the three catalytic dwell positions. As a result, the light intensity of the subsequent power stroke increased from a minimum through a maximum as the AuNR rotated from 0° to 90° relative to that catalytic dwell, then decreased until it reached the next catalytic dwell upon rotating 120°. An arcsine^1/2 function of light intensity (Sielaff et al., 2016; Martin et al., 2018) was used to calculate rotational position versus time. Changes in rotary position versus time (angular velocity) for each power stroke were then averaged and binned to every 3° of rotation, where 0° and 120° refer to catalytic dwell positions.

Examples of V₁ΔHC power strokes that were used to determine the angular velocity profiles versus rotary position are shown in Figure 5. Many V₁ΔHC power strokes rotated continuously (Figure 5A), which are comparable to E. coli F₁ power strokes at saturating MgATP (Martin et al., 2018). However, many other V₁ΔHC power strokes rotated in a faltering manner (Figure 5B) with frequent small oscillations. Some contained a clearly defined dwell during the first 60° of the power strokes (Figure 5C) even in the presence of saturating MgATP, while other power strokes contained a dwell near the end of the power stroke (Figure 5D). Although the power strokes shown were observed in the presence of 5.7 μM MgATP, all four types were present at all three of the MgATP concentrations examined.

The distribution of rotary positions during the power stroke when V₁ΔHC dwells occurred (Figure 6) shows that dwells were most commonly observed ∼45° and 112° after the catalytic dwell. The distribution of the former dwell was significantly broader than the latter, and the peak of the distribution observed at 990, 490, and 5.7 μM MgATP appeared to occur at 45°, 50°, and 40°, respectively (hereafter designated the 45° dwell). This is the approximate rotary position at which F₁-ATPases gives rise to an “ATP-binding” dwell when MgATP is rate-limiting (Yasuda et al., 2001; Bilyard et al., 2013; Martin et al., 2014; Sobti et al., 2021).

The percentage of power strokes examined that contained a 45° dwell increased from 24% at saturating MgATP by 6% and by a further 8% at the rate-limiting MgATP concentrations of 490 and 5.7 μM MgATP, respectively (Table 2). The 184 ± 2 μs average duration of these dwells did not change significantly at the rate-limiting MgATP concentrations. However, the occurrence of the 112° dwell increased by ∼3-fold at 5.7 μM MgATP with respect to that of the 8.5% occurrence observed at saturating MgATP. The duration of these dwells increased by 21% (391 μs) at 5.7 μM MgATP.

The average angular velocity of the V₁ΔHC power stroke vs. rotary position in the presence of saturating (990 μM) MgATP was calculated from the data of 10,274 power strokes examined from 48 V₁ΔHC molecules (Figure 7A). The initial average velocity as the catalytic dwell ended was ∼350°∙ms^–1. The V₁ΔHC decelerated to ∼200°∙ms^–1 at ∼13° (d1-deceleration), then slowly decelerated further from ∼23° to ∼180°∙m^–1 at 60° (d2-deceleration). The rate subsequently accelerated to ∼470°∙ms^–1 at 85° (a1-acceleration), and then rapidly accelerated to briefly reach a rate of ∼1,200°∙ms^–1 at ∼90° (a2-acceleration) before decelerating at ∼93° (d3-deceleration). The rate returned to 300°∙ms^–1 at ∼100° then decelerated to 100°∙m^–1 (d4-deceleration) as it approached the next catalytic dwell at 120°. The V₁ΔHC angular velocity profile was closely similar to that of the E. coli F₁-ATPase (Martin et al., 2018) with the exception that the angular velocity of the latter was significantly slower during the first 70°, and was 21% slower during the spike in velocity at 90° (Figure 7A).

Changes in the angular velocity profile of the V₁ΔHC power stroke vs. rotary position were observed when measured at 490 μM MgATP and at 5.7 μM mgATP (Figures 7B, C), which gave rise to ATPase rates that were 86% and 65% of that observed at 990 μM MgATP, respectively. In the presence of 490 μM MgATP (Figure 7B), the rates after the d2-deceleration, after the a1-acceleration, and after the d3-deceleration were ∼100°∙ ms^–1 at ∼45°, ∼200°∙ ms^–1 at 85°, and ∼180°∙ ms^–1 at 100°. These rates corresponded to rate decreases of 2-fold, 2.4-fold, and 1.7-fold, respectively, from those observed at saturating MgATP. When measured at 5.7 μM MgATP (Figure 7C), the average V₁ΔHC angular velocity profile showed additional decreases in velocity from that observed 490 μM MgATP between rotary positions 60° and 120°. Notably, the 5.7 μM MgATP velocities at 85°, 90°, and 100° were 230°∙ ms^–1, 440°∙ ms^–1, and 110°∙ ms^–1, respectively, which represents decreases of 2.0-fold, 2.7-fold, and 2.7-fold from that observed at saturating MgATP.

The average time required for power strokes to rotate between catalytic dwells were calculated from the angular velocity profiles at each MgATP concentration (Table 1), since each profile reports the time required for each three degrees of rotation. At saturating MgATP (990 μM MgATP), the average power stroke duration was 625 μs while the average power stroke durations at the rate-limiting MgATP concentrations of 490 μM, and 5.7 μM MgATP were 745 and 910 μs, respectively. The power stroke durations at these limiting MgATP concentrations were 1.19-fold and 1.46-fold longer than that measured at saturating MgATP. These increases were comparable to the 1.16-fold and 1.53-fold longer times required to consume an ATP as determined from the ensemble ATPase measurements in the presence of 490 μM and 5.7 μM MgATP, respectively (Table 1). These results support the conclusion that the additional time required for MgATP to bind to the empty catalytic site when MgATP is rate-limiting is evident as a decrease in angular velocity during the power stroke.

The angular velocity profiles (Figure 8) were determined from the average of several thousand power strokes. Consequently, the decreases in average angular velocities observed at limiting MgATP may be due to slower rotation or may occur if a proportion of the power strokes briefly stop rotating, which would be observed as a dwell. The extent that limiting MgATP caused decreases in the angular velocity versus rotary position was determined by taking the difference angular velocity profiles at limiting MgATP from that at saturating MgATP (Figures 8A, B). When compared to the distribution of dwells (black squares) there is a clear correlation between the incidence of dwells and the decreases in angular velocity of the power stroke, but the frequency did not change as MgATP became increasingly limited. This suggests that the 45° dwell does not result from MgATP binding. However, the largest decreases in power stroke angular velocity when MgATP is rate-limiting was observed between 80° and 100° when dwells did not occur. Consequently, these results indicate that the major contribution to the decrease in ATPase rate when MgATP is limiting occurs between 80° and 120°.

The consumption of each ATP requires a consecutive catalytic dwell and power stroke. The power strokes analyzed here are one of three required for a complete rotation of subunit D. Based on the number of power strokes analyzed for each V₁ΔHC molecule during the 5 s data acquisition period, and the average power stroke durations obtained directly from the angular velocity profiles, the average duration of catalytic dwells was determined by subtraction of the time consumed by the power stroke from the total data acquisition time (Table 1). The average catalytic dwell durations in the presence of 990 μM, 490 μM, and 5.7 μM MgATP were calculated to be 7.2 ms, 12.3 ms, and 13.4 ms, respectively. The average durations of the power strokes and catalytic dwells were consistent with the polarizer rotation results that have a minimum time resolution of 1 ms (Figure 4).

The average time required to consume an ATP molecule was also calculated from the sum of the average durations of the power stroke and the catalytic dwells (Table 1). In the presence of 990, 490, and 5.7 μM MgATP, the average times to consume an ATP were calculated from the single-molecule data to be 7.8, 13.0, and 14.3 ms, respectively. It is noteworthy that the times required to consume ATP as measured by the ensemble ATPase assay (Figure 2) at 990, 490, and 5.7 μM MgATP were 105.2, 118.9, and 200 ms (Table 1). These times were considerably longer than those determined by other single-molecule studies, where each molecule was known to be undergoing ATPase-dependent rotation. The ensemble assays reported the average of many V₁ΔHC molecules without knowing how many molecules are actively consuming ATP. By comparing the ensemble and single-molecule results, we estimate that 7%–11% of the V₁ΔHC molecules were actively consuming ATP at any moment in the ensemble assay (Table 1).