Mutation in a non-force-bearing region of protein L influences force-dependent unfolding behavior

Abstract

Abstract Single-molecule magnetic tweezers (MTs) have revealed multiple transition barriers along the unfolding pathway of several two-state proteins, such as GB1 and Csp. In this study, we utilized MTs to measure the force-dependent folding and unfolding rates of both protein L (PLWT) and its Y47W mutant (PLY47W) where the mutation point is not at the force-bearing β-strands. The measurements were conducted within a force range of 3–120 pN. Notably, the unfolding rates of both PLWT and PWY47W exhibit distinct force sensitivities below 50 pN and above 60 pN, implying a two-barrier free energy landscape. Both PLWT and PLY47W share the same force-dependent folding rate and the same transition barriers, but the unfolding rate of PLY47W is faster than that of PLWT. Our finding demonstrates that the residue outside of the force-bearing region will also affect the force-induced unfolding dynamics.