Equilibrium folding and unfolding dynamics to reveal detailed free energy landscape of src SH3 protein by magnetic tweezers

Src SH3 protein domain is a typical two-state protein which has been confirmed by research of denaturant-induced unfolding dynamics. Force spectroscopy experiments by optical tweezers and atomic force microscopy have measured the force-dependent unfolding rates with different kinds of pulling geometry. However, the equilibrium folding and unfolding dynamics at constant forces has not been reported. Here, using stable magnetic tweezers, we performed equilibrium folding and unfolding dynamic measurement and force-jump measurement of src SH3 domain with tethering points at its N- and C-termini. From the obtained force-dependent transition rates, a detailed two-state free energy landscape of src SH3 protein is constructed with quantitative information of folding free energy, transition state barrier height and position, which exemplifies the capability of magnetic tweezers to study protein folding and unfolding dynamics.