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...

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.