Temperature Dependence of the Mechanical Unfolding of Single Ubiquitin Proteins

Abstract

Single molecule atomic force microscopy (AFM) in force-clamp mode was used to study the effect of temperature on the mechanical unfolding of polyubiquitin. Single protein chains were extended at a constant force and the unfolding of individual domains was measured from the staircase increase of the contour length. The unfolding rate constant at each pulling force and medium temperature was obtained from the exponential fit to the ensemble averages of the contour length traces. The unfolding rate at zero force and the distance to transition state were then calculated from the force-dependent rate constants. By varying the temperature in the 5-45 °C range and the pulling force in the 70-210 pN interval, we find a significant thermal effect on unfolding. Fitting the temperature dependant unfolding rate at zero force k to the Arrhenius equation k =A exp(-Ea/kT) yields an activation energy Ea of 87 ± 5 kJ/mol and an exponential pre-factor A ∼ 2.3·1012 s−1. The exponential pre-factor yields a similar value to predictions of statistical mechanic calculations derived by the transition state theory (TST), of (5.8-6.6)·1012 s−1 for the considered temperatures. This correlation suggests that the unfolding of proteins is not subject to frictional effects and that all interactions with the solvent molecules favor the unfolding, independent of the collision angle. This finding will prove important in assessing the energy landscape of unfolding proteins as a transition state process.