Effect of ssDNA ligand in modulating the folding and unfolding dynamics of cold shock protein BcCsp

The cold shock domain (CSD) protein acts as a nucleic acid-binding protein and exhibits a strong affinity for the Y-box core motif ATTGG segment on gene promoter regions, playing a significant role in gene regulation. Given the moderate folding and unfolding rates of the cold shock protein from Bacillus caldolyticus (BcCsp) observed in previous magnetic tweezers studies, it is a suitable choice for investigating the interaction between CSD protein and single-stranded DNA containing ATTGG. While earlier studies utilizing single molecule force spectroscopy delved into the mechanical characteristics of BcCsp, the impact of ssDNA on its folding and unfolding dynamic behaviors has yet to be investigated. Here, utilizing stable magnetic tweezers, we compared the force-dependent folding and unfolding rates of BcCsp in the presence of different concentrations of TGATTGG ssDNA. We found that folding rate of BcCsp keeps the same, while unfolding rate decreases as the concentration of TGATTGG increases. Detailed unfolding free energy landscape analysis revealed that ssDNA not only binds to the native state of BcCsp, but also binds to the partially unfolded conformation to change the unfolding barrier. Our study demonstrates the coupling between protein folding or unfolding and ligand association/dissociation dynamics.