Figure 1.

(A) Schematic representation of the measurement apparatus. A magnetic bead is tethered to a glass capillary by a single, nicked DNA molecule. Magnets above the capillary create a field gradient that pulls on the bead with a force F, which is determined by measuring the bead's lateral fluctuations dx. By optically tracking the bead, we measure the extension L of the DNA with time. (B) Typical measurement of DNA extension, both with and without FtsK_50C in the solution. The plotted data were measured at F=10.7 pN and with 5 mM ATP; the data sets have been offset for clarity. We attribute the transient decreases in DNA extension to the creation of loops of DNA by single complexes of FtsK_50C. (C) A typical individual event extracted from the data shown in (B). All events begin with a constant-velocity decrease in DNA extension; we attribute the slope of the descent to the translocation velocity of the complex. The velocity, distance travelled d, and on-time are easily measured because of the well-defined event shape. As sketched, loop formation requires the complex to bind the DNA in two locations; see Figure 6 and Discussion for details. (D) FtsK_50C activity versus ATP concentration for both bulk (rate of ATP consumption; red curve) and single molecule (translocation velocity; green points) assays. Both data sets are well fit by the Michaelis–Menten equation V_max[ATP]/([ATP]+K_m), with, respectively, V_max=30±2 ATP/s and 6.7±0.1 kbp/s, and K_m=270±40 and 330±20 μM. The fit to the single-molecule data (blue curve) is shown; it is highly coincident with the bulk data fit, which is omitted for clarity. Single-molecule data are taken at F∼5 pN, and each point is the average of typically 100 events; plotted bars indicate standard error. Inset: Histogram of measured translocation velocities (open circles) with best-fit Gaussian curve (solid line); data taken at 5 mM ATP and F=6 pN.