Dynamic force sensing of filamin revealed in single-molecule experiments

Fig. 2.

Interaction of filamin with tethered peptides. (A) Force extension trace of the interaction between the target peptide of GPIbα and FLNa21 (GPIbα-FLNa21). At forces around 10 pN, the target peptide rapidly fluctuates between bound and unbound conformations and stays permanently unbound at higher loads (>12 pN). (B) Equilibrium traces obtained at three different biasing forces. At high loads (top trace), the probability (black histogram) is shifted to the unbound state, whereas at decreasing loads, the bound state becomes more and more populated (bottom two traces). (C) Opening (circles) and closing (triangles) rates as a function of force. The solid line is an extrapolation of the rates to zero-load taking into account the compliance of all mechanical elements in the construct (SI Materials and Methods). (D) Force extension trace of the interaction between the target peptide of β7-integrin and FLNa21 (ITβ7-FLNa21). (E) Equilibrium traces of ITβ7-FLNa21 obtained at three different biasing forces (compare B). (F) Opening (circles) and closing (triangles) rates as a function of force (compare C). (G) Force extension trace of the interaction between the target peptide of migfilin and FLNa21 (Mig-FLNa21). (H) Equilibrium traces of Mig-FLNa21 obtained at three different biasing forces (compare B and E). (I) Opening (circles) and closing (triangles) rates as a function of force (compare C and F).

Fig. 3.

Single-molecule mechanical competition assay to study peptide binding from solution. (A) Time traces of opening and closing of the GPIbα-FLNa21 construct held at a force bias of 8.5 pN in the presence of 2.3 μM GPIbα peptide in solution. The colored traces correspond to the 20-kHz data (gray), moving average filtered with 2.5 ms (black) and 50 ms (white) time window. Apparent high-SD regions with lifetimes

in the black and gray traces are interrupted by low-SD regions with lifetimes . (B) A zoom into the blue region shows the transition between a high-SD (rapid opening and closing cycles of the tethered construct) and low-SD region (blocked fluctuations due to competitive peptide binding from solution). (C) Dependence of the bound and unbound lifetimes as a function of applied force and solution concentration. As expected for binding from solution, depends on the opening probability of the tethered construct and hence the applied force, as well as the solution concentration, whereas is independent (see text and SI Materials and Methods).

Fig. 4.

Filamin domain pairs act as a precisely tuned mechano-sensor. (A) Low-resolution stretch (blue) and relax (gray) trace of FLNa20-21. At forces around 15 pN, FLNa20 unfolds, and at higher loads exceeding 30 pN, FLNa21 unfolds. Opening of the domain pair and release of the autoinhibition is not visible at this experimental resolution. (B) (Upper) Competition assay of domain pair opening in the presence of 2.7 μM GPIbα peptide in solution observed at loads of 4.5 pN (color scheme as in Fig. 3A). High-SD regions (opening–closing fluctuations) are interrupted with low-SD regions where bound peptide blocks fluctuations. (Lower) Zoom into a transition region. (C) Force-dependent binding of peptide from solution observed at biasing forces from 2.3 to 4.0 pN. From low to high loads, the binding probability (black histograms) increases constantly. (D) Force-dependent gating characteristics of the force-sensing domain pair as obtained from the force and concentration-dependent dwell times of Fig. S4C (blue data points; black solid line shows global fit). The three symbols (triangle, square, and circle) denote three different solution concentrations. The dashed black line is an independent measure of the force-dependent opening probability as obtained from the HMM analysis of the fluctuating state where no ligand from solution is bound (Fig. S6B).