Structure-function dynamics of protein, as a flexible polymer, is essential to describe their biological functions. Here, using a single-molecule magnetic tweezers, we have studied the effect of ionic strength on...

Studies of free energy, kinetics or elasticity are common to most disciplines of science. Detailed quantification of these properties demands number of specialized technologies. Furthermore, monitoring 'perturbation' in any of these properties, in presence of external stimuli (protein/DNA/drugs/nanoparticles etc.), requires multiple experiments. However, none of these available technologies can monitor these perturbations simultaneously in real time on the very same molecule in a single shot experiment.Here we present real-time microfluidics-magnetic tweezers technology with the unique advantage of tracking a single protein dynamics for hours, in absence of any significant drift, with the flexibility of changing physical environment in real time. Remarkable stability of this technique allows us to quantify five molecular properties (unfolding kinetics, refolding kinetics, conformational change, chain flexibility, and ∆G for folding/unfolding), and most importantly, their dynamic perturbation upon interacting with salt on the same protein molecule from a single experiment. We observe salt reshapes the energy landscape by two specific ways: increasing the refolding kinetics and decreasing the unfolding kinetics, which is characterized as mean first passage time. Importantly, from the same trajectory, we calculated the flexibility of the protein polymer, which changes with salt concentration and can be explained by our modified 'electrolyte FJC model'. The correlation between ∆G, kinetics and polymer elasticity strongly argues for a stiffness driven energy landscape of proteins. Having the advantage of sub nanometer resolution, this methodology will open new exciting window to study proteinsone such examples is demonstrated in this article: electrolyte driven conformational fluctuation under force, which was not studied before.

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