Comparative analysis of folding and unfolding dynamics and free-energy landscapes in homologous cold shock proteins with variable thermal stabilities

Abstract

Cold shock proteins from the mesophilic organism (BsCSP), the thermophilic organism (BcCSP), and the hyperthermophilic organism (TmCSP) are homologous proteins with similar native structure but quite different thermal stabilities, which makes them promising candidates for investigating the general and specific aspects the folding and unfolding dynamics of proteins and free-energy landscapes. In this study, we employed magnetic tweezers to explore the force-dependent folding and unfolding rates of BsCSP, BcCSP, and TmCSP at forces from several pN to tens of pN. Our results indicate similar force-dependent folding rates for these three proteins, while the unfolding rate of BsCSP, BcCSP, and TmCSP decreases sequentially, consistent with previous biochemical findings. Comparing with TmCSP, BcCSP shows lower but sufficient stability with folding free energy of 8.0 kBT, and its force-dependent unfolding rates exhibit tremendous nonlinear behavior deviated from Bell's model like TmCSP. BsCSP has very low stability with folding free energy of only 3.9 kBT, and its force-dependent unfolding rates at 3–10 pN exhibit almost linear behavior. Comprehensive free-energy landscapes are constructed, revealing common barriers that governs folding and unfolding dynamics for BcCSP and TmCSP but a wide merged barrier for BsCSP. Our findings provide valuable insights into the folding and unfolding mechanisms of these CSPs, shedding light on the relationship between protein structure, stability, and mechanical properties. Published by the American Physical Society 2024