Microsecond resolution of single-molecule rotation catalyzed by molecular motors

Abstract

Single-molecule measurements of rotation catalyzed by the F(1)-ATPase or the F(o)F(1) ATP synthase have provided new insights into the molecular mechanisms of the F(1) and F(o) molecular motors. We recently developed a method to record ATPase-driven rotation of F(1) or F(o)F(1) in a manner that solves several technical limitations of earlier approaches that were significantly hampered by time and angular resolution, and restricted the duration of data collection. With our approach it is possible to collect data for hours and obtain statistically significant quantities of data on each molecule examined with a time resolution of up to 5 μs at unprecedented signal-to-noise.

Fig. 1

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. Each histogram contains 3,520 measurements at each position of the polarizer obtained with the data acquisition speeds used to collect data points for c-ring rotation. The polarizer was then rotated counterclockwise by 10° and data collection was repeated.

Fig. 2

Schematic of the dark-field microscope used to make the measurements. In this configuration, the scattered light from a single rotating gold nanorod (dashed line) passes through a beam splitter for measurement by two single photon counting avalanche photodiode detectors when the orientation of the polarizer for detector 1 is offset from that of detector 2.

Fig. 3

(a) Assemby of FOF1nanodiscs (n-F F) with a gold nanorod on a microscope slide for single molecule studies. Microscope slide bound n-F Fattached via β-subunit N terminus 6xHis tags attached to an avidin-coated 77 × 39 nm nanorod via a biotinylated subunit-c cys that was inserted as the second residue in the sequence. Integral membrane protein subunits of F are incorporated into a nanodisc comprised of the membrane scaffold protein (MSP) that forms a 13-nm diameter ring of a-helices around a bilayer of a few hundred phospholipid molecules, and has been shown to provide a good model for lipid bilayers. Rotation catalyzed by purified F -ATPase is measured by assembling F on the microscope slide in the same manner, but the Avidin-coated gold Nanorod is attached to a biotinylated cys on the γ-subunit not shown). (b) Dwell duration and positions for ATPase-driven F rotation using data acquired at 1 kHz. Horizontal lines indicate the catalytic dwells.

Fig. 4

Relationship between a 120° power stroke and a 90° measured rotational transition. Theoretical plot of the intensity of scattered red light from a nanorod during one complete revolution that involves three consecutive power strokes and three consecutive catalytic dwells separated by exactly 120°. The nanorod is initially positioned almost, but not exactly perpendicular to the orientation of the polarizer such that the scattered light intensity goes through a minimum than a maximum prior to catalytic dwell 1. A transition includes the data between the minimum and maximum intensities representing 90° of the 120° of rotation for analysis. When initial alignment of the nanorod is exactly at the minimum and each of the successive power strokes is exactly 120° the algorithm selects transitions for power strokes 1 (min to max) and 3 (max to min).

Fig. 5

Power stroke events with and without transient dwells due to ATPase-driven rotation of single molecules. Example transitions for F₁ (filled circle), as well as for n-F_oF₁ with (filled square) and without (open square) transient dwells. Arrows indicate transient dwells. Nanorod attachment occurred via the γ-subunit for F₁ or via the c-ring for n-F_oF₁. Data were acquired at 100 kHz in the presence of 15% PEG400 (v/v) and 1 mM MgATP, and were converted from scattered light intensity to degrees of rotation by Eq. 1.

Fig. 6

Transition times of F₁-dependent γ-subunit rotation as a function of PEG-400 concentration and nano-rod size at 1 mM MgCl₂ and 2 mM ATP. (a) Average transition times as a function of PEG400 concentration measured using 75 × 35 nm (triangle), 87 × 36 nm (open circle), 90 × 46 nm (square) and 91 × 45 nm (diamond) nanorods. (Inset) Expansion of data between 0 and 20% PEG400 shows that the transition times for the three smallest nanorods converge to a single value of ~250 μs. (b) Transition times plotted as a function of increasing nanorod size at fixed PEG400 concentrations. Lines are the linear best fit for 30, 45, and 60% PEG400.

Fig. 7

(a) Theoretical plot of changes in intensity of red scattered light observed from two single rotating nanorod by two photon counters when the orientation of the polarizer for detector 1 is offset from that of detector 2. (b) Experimental time course of rotational position of the γ subunit in a single F₁ molecule determined from data acquired at 10 kHz with two photon counters in which the orientation of the polarizer for detector 1 was offset from that of detector 2. The substrate concentration was 1 mM MgCl and 2 mM ATP. (c) Detail of the rotational stepping of the boxed region of (b). Horizontal lines indicate the 120° catalytic dwell positions.