Edited by Taekjip Ha, Johns Hopkins University, Baltimore, MD, and approved February 21, 2019 (received for review October 4, 2018)
High-speed and MD force spectroscopy of SA–b unbinding. (A, Left) HS-FS setup. SA agarose beads (Top Left) were immobilized on the sample surface while b was covalently attached to the microcantilever (Bottom Left) through a PEG linker (contour length ∼10 nm). (A, Right) MD simulations setup and the SA–b tetramer used in the MD simulations with labeled loops 3–4 and 7–8 (PDB ID code 3RY2). (B) Experimental force–distance traces at velocities from 1 µm/s to 30,556 µm/s revealing bond rupture events using AC7 (top curve) and AC10 cantilevers (bottom curves). The stretching profile of the experimental curves was the result of stretching the PEG linker and deforming the agarose bead to which SA was linked. (C) Force–extension traces from the MD simulations at retraction velocities from 1,000 µm/s to 2 × 106 µm/s.
Dynamic force spectrum of SA/b unbinding. Most probable rupture forces (± SEM) from HS-FS experiments (circles, using regular AC10 and fast AC7 cantilevers, cyan and magenta, respectively) and MD simulations (triangles). The blue line represents a Brownian dynamics fit to the whole force spectrum. (Inset) The resulting equilibrium free-energy landscape is shown as a blue line, which revealed two transition barriers (with parameters D = 4 × 107 nm2/s, ΔG1 = 17 kBT, ΔG2 = 21 kBT, xβ1 = 0.19 nm, and xβ2 = 0.44 nm) and a possible intermediate state between the two (blue dashed line). The red dashed line sketches a third, outer barrier (Fig. 3 and text), which cannot be extracted from the dynamic force spectrum.
Outer barrier. (A) Distribution of COM distances between SA and b for all MD trajectories during unbinding. The first two minima coincide with the barrier positions obtained from the Brownian dynamics simulations fit of the force spectrum in Fig. 2, Inset. (B) Examples of MD and (C, top three) HS-FS force–time curves showing outer binding. The lifetime and applied force of outer binding events was determined as shown (red lines) and described in Materials and Methods. The bottom curve shows an example for which no intermediate was observed. The red line shows an exponential fit with decay time ∼0.28 µs. (D) Distribution of experimental distance to outer binding (left axis) and pulling velocity dependence of the average distance (blue, right axis). (E) Outer binding lifetime vs. applied force from HS-FS (circles) and MD simulations (triangles). The solid line shows the best fit to the force-dependent lifetime DHS model (14) with parameters τ0 = 16 ± 7 µs, xβ = 0.16 ± 0.10 nm, and ΔG = 12 ± 6 kBT. This distance the outer barrier (xβ) should be added to the position of the second barrier at xβ2 = 0.60 nm in Fig. 2 (sketched as a dashed red line in the inset).
Dynamic multiplicity of SA–b unbinding pathways. (A) Bound and intermediate states with unbinding pathways observed during forced dissociation in MD trajectories. The line color reflects pulling velocity from slow (blue) to fast (red) and thickness reflects passage frequency. b molecular representations show overlays of the hydrogen bonds with SA residues for the four intermediate states. The amino acids with the strongest intermediate interactions are labeled in red. (B) The energy of the H-bonds between b and the most important residues is shown below as a function of the COM distance. (C) Structural snapshots of the different intermediate states showing H-bonds between SA residues and b.
