Subangstrom single-molecule measurements of motor proteins using a nanopore

Abstract

Techniques for measuring the motion of single motor proteins, such as FRET and optical tweezers, are limited to a resolution of ∼300 pm. We use ion current modulation through the protein nanopore MspA to observe translocation of helicase Hel308 on DNA with up to ∼40 pm sensitivity. This approach should be applicable to any protein that translocates on DNA or RNA, including helicases, polymerases, recombinases and DNA repair enzymes.

Figure 1. The nanopore system

(a) A phospholipid lipid bilayer (purple) spans a Teflon (light blue) aperture, separating a KCl solution into cis and trans compartments. A single MspA nanopore (gold) is inserted into the bilayer. A voltage applied via two Ag/AgCl electrodes causes ion current through the pore. The electric potential also attracts enzyme-DNA complexes into the pore (green and black; not to scale). (b) ssDNA bound to the motor enzyme (polymerase or helicase) threads through the pore’s constriction until the enzyme comes to rest on the pore rim. The enzyme controls the DNA’s motion through the pore, while the nucleotides positioned within MspA’s constriction govern the ion current. (c) The phi29 DNA polymerase (DNAP) moves the DNA through MspA in single-nucleotide steps resulting in distinct current levels. Black lines mark the average current of observed levels. Breaks in the current trace are for current levels lasting more than 200 ms. Back-stepping of the phi29 DNAP causes repetitions of levels, indicated by *. (d) The mean ion current of the time-ordered levels and overlay the pattern of current levels for 31 recordings of the same sequence of DNA. The associated DNA sequence is shown; ’X’ is an abasic residue. (e) Zoomed in view of the dashed box in (d) demonstrates conversion of current measurement to DNA position using a smooth curve (spline) fit to the current pattern. The standard deviation translates to uncertainty in DNA position is as low as 0.06 nt. (f) Illustration showing that lower voltage, i.e. decreased force, reduces the DNA’s elongation and shifts its position within MspA’s constriction. (g) Comparison of ion current levels recorded at 180 mV (black circles) and at 140 mV (green triangles). Peaks of the spline interpolation illustrate a shift of the DNA’s position. (h) Current values for 180 mV (black circles) and a spline interpolation to those levels (black curve). Green triangles show the current levels taken at 140 mV in panel (c) after applying a multiplicative scale and additive current offset. The scaled 140 mV levels were horizontally displaced by δ = 0.29 nt to put them in line with the 180 mV spline. “Level number” refers to the number assigned to each level as it appears in order while “DNA position” refers to the position of DNA within the pore. We define integer DNA positions to be identical to phi29 DNAP level numbers.

Figure 2. SPRNT applied to the helicase Hel308

(a) Consensus of current level patters for 20 reads of DNA “sequence A” with phi29 DNAP controlling DNA translocation through the pore. (b) Same as in a except for 72 reads using Hel308 to control the DNA motion. (c) Means of current levels recorded with Hel308 actuated DNA movement (orange and blue symbols) scaled to match a spline (grey curve) of the levels found with phi29 DNAP-controlled movement (black points), also shown in (a). The shaded levels in (b), indicated with orange diamonds, were similar to levels found with phi29 DNAP but were horizontally offset by −0.14 nt in order to best match the spline of levels taken with phi29 DNAP. The unshaded levels in (b), indicated with blue circles, were offset by + 0.41 nt relative to the single nucleotide step positions taken by phi29 DNAP. (d) Expanded view of DNA positions 25 through 27, with colors indicating the same elements as (c). As in Figures 1e, h, we illustrate the use of the spline of the phi29-DNAP levels as a distance scale to find the position of even and odd numbered levels found with Hel308 (Supplemental Discussion 2). (e) Median duration of corresponding current levels in (b) for two different ATP concentrations: 10 µM (blue) and 1 mM (red) ATP. The median duration of shaded levels is dependent on [ATP]. (f) The ratio of the median durations with high and low [ATP] removes sequence dependence that also influences the step durations (Supplemntal discussion 4). The levels alternate between ATP-independent levels (marked with blue dots) and ATP-dependent levels (marked with orange diamonds).