Connecting conformational stiffness of the protein with energy landscape by a single experiment

Abstract

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.

Direct observation of the mechanical role of bacterial chaperones in protein folding

“…The changes in length between the centers of two histogram peaks denotes the step size. Detailed information regarding RT-MMT techniques and image processing are described in our previous article 12 . The magnetic tweezers experiment was carried out with 5 nM of protein L in a buffer containing 20mM sodium phosphate, 140 mM NaCl and 10mM ascorbic acid and pH of 6.8.…”

“…The changes in length between the centers of two histogram peaks denotes the step size. Detailed information regarding RT-MMT techniques and image processing are described in our previous article 12…”

“…To address this question, we investigated the mechanical role of different chaperones and chaperone-like proteins under force, individually and in combinations: DnaK, DnaJ, SecB, DsbA, Trigger factor (TF), protein disulfide isomerase (PDI) and thioredoxin (Trx). The mechanical effects of these chaperones were monitored by our novel real-time microfluidic-magnetic-tweezers (RT-MMT) that implements both force clamp and force ramp technology 12 . Using the force clamp technology constant force can be provided on a protein, offering a unique way to probe both thermodynamics and kinetic properties of the chaperone-protein interactions under equilibrium condition.…”

Real time observation of chaperone-modulated talin mechanics with single molecule resolution

“…Details information regarding the image processing and techniques are described previously. 18 RT-MMT experiment was performed with 1-10 nM of protein in a buffer containing 140 mM NaCl, 20 mM sodium phosphate and 10 mM ascorbic acid. 41,42 Ascorbic acid was used as antioxidant for preventing the oxidative damage of the protein.…”

“…To address this question, single molecule real-time microfluidics magnetic tweezers (RT-MMT) has been used, which executes both force ramp and force clamp technologies. 18 The force ramp technology, by increasing or decreasing the force at a constant rate, probes unfolding and refolding events of a protein; while applying the force clamp technology, a constant force can be applied to the protein, allowing to detect their thermodynamics and kinetic properties under equilibrium condition. Owing to the advantage of wide force regime of 0-120 pN with sub-pN resolution, we were able to observe chaperone interaction both at folded and unfolded state, independently.…”