Talin as a critical focal adhesion mechanosensor exhibits force-dependent folding dynamics and concurrent interactions. Being a cytoplasmic protein, talin also might interact with several cytosolic chaperones; however, the roles of chaperones in talin mechanics remain elusive. To address this question, we investigated the force response of a mechanically stable talin domain with a set of well-known unfoldase (DnaJ, DnaK) and foldase (DnaKJE, DsbA) chaperones, using single-molecule magnetic tweezers. Our findings demonstrate that chaperones could affect adhesion proteins’ stability by changing their folding mechanics; while unfoldases reduce their unfolding force from ~11 pN to ~6 pN, foldase shifts it upto ~15 pN. Since talin is mechanically synced within 2 pN force ranges, these changes are significant in cellular conditions. Furthermore, we determined that chaperones directly reshape the energy landscape of talin: unfoldases decrease the unfolding barrier height from 26.8 to 21.7 kBT, while foldases increase it to 33.5 kBT. We reconciled our observations with eukaryotic Hsp70 and Hsp40 and observed their similar function of decreasing the talin unfolding barrier. Quantitative mapping of this chaperone-induced talin folding landscape directly illustrates that chaperones perturb the adhesion protein stability under physiological force, thereby, influencing their force-dependent interactions and adhesion dynamics.

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...

Protein folding under force is an integral source of generating mechanical energy in various cellular processes, ranging from protein translation to degradation. Although chaperones are well known to interact with proteins under mechanical force, how they respond to force and control cellular energetics remains unknown. To address this question, we introduce a real-time magnetic tweezer technology herein to mimic the physiological force environment on client proteins, keeping the chaperones unperturbed. We studied two structurally distinct client proteins––protein L and talin with seven different chaperonesindependently and in combination and proposed a novel mechanical activity of chaperones. We found that chaperones behave differently, while these client proteins are under force, than their previously known functions. For instance, tunnel-associated chaperones (DsbA and trigger factor), otherwise working as holdase without force, assist folding under force. This process generates an additional mechanical energy up to ∼147 zJ to facilitate translation or translocation. However, well-known cytoplasmic foldase chaperones (PDI, thioredoxin, or DnaKJE) do not possess the mechanical folding ability under force. Notably, the transferring chaperones (DnaK, DnaJ, and SecB) act as holdase and slow down the folding process, both in the presence and absence of force, to prevent misfolding of the client proteins. This provides an emerging insight of mechanical roles of chaperones: they can generate or consume energy by shifting the energy landscape of the client proteins toward a folded or an unfolded state, suggesting an evolutionary mechanism to minimize energy consumption in various biological processes.

DsbA is a ubiquitous bacterial oxidoreductase that associates with substrates during and after translocation, yet its involvement in protein folding and translocation remains an open question. Here we demonstrate a...

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.

Force is a crucial protein denaturant in cells, occurring during processes including translation, degradation, and translocation. In bacteria, many toxins and virulence factors pass through a translocon pore as unfolded polypeptides en route to periplasmic and extracellular compartments.DsbA is a ubiquitous bacterial oxidoreductase that associates with substrates during and after translocation, yet its involvement in protein folding and translocation remains an open question.Here, using magnetic tweezers-based single molecule force spectroscopy, we characterize a mechanical chaperone activity of DsbA on a cysteine-free domain from the protein L superantigen. Interaction of DsbA with unfolded protein L substrates prominently increases the fraction of time that protein L spends in its native folded state. This chaperone activity is tuned by the oxidation state of DsbA; oxidized DsbA is a strong promoter of folding, but the effect is weakened by reduction of the catalytic CXXC motif. We further localize the chaperone binding site of DsbA using a seven residue peptide which effectively blocks the chaperone activity. We calculated that DsbA assisted folding of proteins in the periplasm generates enough mechanical work to decrease the ATP consumption needed for periplasmic translocation by up to 33%. In turn, pharmacologic inhibition of this chaperone activity may open up a new class of antivirulence agents.

Protein folding under force is an integral source of generating mechanical power to carry out diverse cellular functions. Though chaperones interact with proteins throughout the different stages of folding pathways, how they behave and interact with client proteins under force was not known. Here we introduce the ‘mechanical role’ of chaperone and explained it with seven independent chaperones using single molecule based real-time microfluidics-magnetic-tweezers. We showed and quantified how chaperones increase or decrease mechanical work output by shifting the folding energy landscape of the client proteins towards the folded or unfolded state. Notably, we found chaperones could behave differently under force. For instance: trigger factor, ribosomal-tunnel associated chaperone, working as a holdase in absence of force, but assist folding under force. This phenomenon generates extra mechanical energy to pull the polyprotein from the stalled ribosome. This is also relevant for SecYEG tunnel associated oxidoreductase DsbA, which act similarly like TF and increases the mechanical energy up to ~59 zJ, to facilitate membrane translocation in an energy efficient manner. However cytoplasmic oxidoreductases such as PDI and Thioredoxin, unlike DsbA, do not have the mechanical folding ability. Interestingly, we observed a highly potential foldase- DnaKJE chaperone complex, only restores the folding ability of the client protein and fails to act like TF or DsbA under force. However, the individual components of this complex, DnaK or DnaJ, act as a mechanical holdase and inhibits folding; similar to that of SecB. Together our study provides an emerging insight of mechanical chaperone behavior, where tunnel associated chaperones generate extra mechanical work whereas the cytoplasmic chaperones are unable to generate that, which might have evolved to minimize the energy consumption in biological processes.

Recent single molecule studies have recognized talin as a mechanosensitive hub in focal adhesion, where its function is strongly regulated by mechanical force. For instance, at low force (less than 5pN), folded talin binds RIAM for integrin activation; whereas at high force (more than 5pN), it unfolds to activate vinculin binding for focal adhesion stabilization. Being a cytoplasmic large protein, talin must interact with various chaperones, however the role of chaperones on talin mechanics is unknown. To address this question, we investigated the force response of a mechanically stable talin domain, with a set of well-known holdase and foldase chaperones, using a single molecule magnetic tweezers technology. Our findings demonstrate a novel mechanical role of chaperones. We found holdase chaperones reduce the mechanical stability of the protein to ~6 pN, while the foldase chaperone increases it up to ~15 pN. The alteration in mechanical stability ascribes to the underlying molecular mechanism where the chaperones directly reshape the energy landscape of talin. For example, unfoldase chaperone (DnaK) decreases the unfolding barrier height from 26.8 to 21.69 kBT and increases the refolding barrier from 3.49 to 11.31 kBT. In contrast, foldase chaperone (DsbA) increases the unfolding barrier to 33.46 kBT and decreases the refolding barrier to 0.44 kBT. The quantitative mapping of the chaperone-induced free energy landscape of talin directly shows that chaperones could perturb the focal adhesion dynamics, which in turn can influence downstream signaling cascades in diverse cellular processes.