Single Molecule Unzipping of Coiled Coils: Sequence Resolved Stability Profiles

We use a high resolution atomic force microscopy technique to mechanically unzip and rezip single coiled-coil proteins. This allows us to read off the complete stability profile of the protein turn by turn. We investigated three coiled coils with different length as well as a point mutation and find force fluctuations between 9 and 15 pN that can be directly related to the amino-acid sequences. An equilibrium model previously applied to DNA fully describes the mechanical unzipping process including free-energy contributions of the individual turns and seed formation energy.

Figure 1

(a) Schematics of the experimental single-molecule setup for unzipping a double-stranded coiled coil. Binding of the ddFLN1-5 domains (beads) to substrate and tip is unspecific and does not necessarily occur at ddFLN1. DdFLN4 is shown as a black square. The ladder illustrates the coiled-coil construct. (b) Representative force-extension trace of the ddFLN1-5-LZ26 construct. The sawtooth pattern arises from ddFLN1-5 domain unfolding. The unfolding events of ddFLN4 are marked with squares. In each force trace the molecule was stretched and relaxed consecutively up to 14 times in an extension range between 20 and 140 nm since this is the expected range for coiled-coil unzipping. (c) To increase signal-to-noise ratio those stretching traces (crosses) and relaxation traces (circles) were averaged separately. Around 12 pN specific force fluctuations are visible reflecting the coiled coil unzipping and rezipping behavior. (d) A zoom into the force fluctuation pattern with further increased signal-to-noise ratio obtained by averaging five traces from different molecules.

Figure 2

Unfolding (crosses) and folding (circles) force-extension traces of the three investigated coiled coils compared to the theory. The mean pulling velocities are 460, 510, and $690\mathrm{nm}/\mathrm{s}$ for the LZ10, LZ18, and LZ26 coiled coils, respectively.

Figure 3

Calculated force-extension trace of the LZ18 coiled coil (solid line) compared to the data (crosses) as in Fig. 2. Overlaid is the calculated force-extension trace of a LZ18 mutant where the asparagine in turn five was replaced by a valine (dotted line) and the experimental data (quadrates). The difference between the force-extension traces from both zippers are localized at the $n$ terminus while the $c$-terminal force-extension behavior is not affected.