The Influence of DNA Handles on the Mechanical Response of Single Protein Molecules.

The mechanical response of proteins to force is governed by their chain stiffness, molecular length, and domain segmentation and can be influenced by unstructured tethers in series with the molecule. Here, we investigate the effect of DNA linkers on the mechanical unfolding of proteins. These tethers are extensively used in single-molecule techniques as spacing handles or calibration standards. We designed two DNA-protein constructs made from covalently cross-linked DNA molecules having 604 bp and 3 kbp in series with eight repeats of bacterial protein L, and compared them with the protein L construct lacking any DNA linker. Using magnetic tweezers, we measured the unfolding dynamics and folding likelihood of protein L connected in series with these DNA linkers. Our findings indicate that stiff DNA linkers do not significantly alter the unfolding kinetics of the tethered protein, while a longer handle slightly increases the force required for refolding. We rationalize our measurements using an energy profile model projected on the pulling end-to-end reaction coordinate. Furthermore, we analyze how the tension is being transmitted along the protein-DNA construct as a function of its size. We conclude that the small differences induced by the presence of DNA linkers in single-molecule measurements are insignificant, given the current instrumental capabilities.