Fig. 1.
Experimental design for time resolved measurements of the recoil dynamics of an extended polypeptide. (A) Schematic diagram of the force-clamp apparatus. A single polypeptide is extended between a high-speed cantilever (Olympus biolever) and a piezoelectric actuator with a high resonant frequency (300 kHz, Physik Instrumente PL-055.30). A well-tuned PID amplifier adjusts the piezoelectric actuator so as to keep the force on the polypeptide at the set point value (Fsp). Under force clamp conditions, Fc = Fpol + FD, where Fc is the force applied by the cantilever, Fpol is the elastic force produced by the molecule and FD is the flow drag force (dashed lines) from the motion of the piezoelectric actuator. (B) Typical experimental trace of the end-to-end length corresponding to the force protocol on the lower box. The inset shows a staircase of ubiquitin unfolding events (stars) measured at 180 pN providing a strong fingerprint for a single polypeptide and measures its initial length. The extended polypeptide is then repeatedly cycled between 250 pN and 100 pN in order to collect many trajectories (7 < n < 30) until the molecule detaches. (C) Example of a recoil trajectory (blue) obtained by averaging eight consecutive recoil trajectories. The averaged force trace shows a step (250 pN down to 100 pN) with a half time of approximately 33 μs, which is more than 10 times shorter than the relaxation time constant of the polypeptide measured to be approximately 1.11 ms. After recoil, the corresponding extension (green trace) is much faster with a time constant of approximately 0.68 ms. The force traces are superimposable. (D) Potential of mean force of a 250 nm long polymer calculated at two pulling forces of 250 pN and 100 pN, using the WLC model of polymer elasticity (lower box). The figure highlights the physics of our experimental design. The polypeptide starts extended at 250 pN (1) and then it is abruptly relaxed to 100 pN (2) where it recoils down to its new minimum (3), then it is switched back to the high force (4) where it extends to the PMF minimum at 250 pN (1).