RESUMEN

Osmolytes are well known to protect the protein structure against different chemical and physical denaturants. Since their actions with protein surfaces are mechanistically complicated and context dependent, the underlying molecular mechanism is not fully understood. Here, we combined single-molecule magnetic tweezers and molecular dynamics (MD) simulation to explore the mechanical role of osmolytes from two different classes, trimethylamine N-oxide (TMAO) and trehalose, as mechanical stabilizers of protein structure. We observed that these osmolytes increase the protein L mechanical stability by decreasing unfolding kinetics while accelerating the refolding kinetics under force, eventually shifting the energy landscape toward the folded state. These osmolytes mechanically stabilize the protein L and plausibly guide them to more thermodynamically robust states. Finally, we observed that osmolyte-modulated protein folding increases mechanical work output up to twofold, allowing the protein to fold under a higher force regime and providing a significant implication for folding-induced structural stability in proteins.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

The structure-function dynamics of a protein as a flexible polymer is essential to describe its biological functions. Here, using single-molecule magnetic tweezers, we have studied the effect of ionic strength on the folding mechanics of protein L, and probed its folding-associated physical properties by re-measuring the same protein in a range of ammonium sulfate concentrations from 150 mM to 650 mM. We observed an electrolyte-dependent conformational dynamics and folding landscape of the protein in a single experiment. Salt increases the refolding kinetics, while decreasing the unfolding kinetics under force, which in turn modifies the barrier heights towards the folded state. Additionally, salt enhances the molecular compaction by decreasing the Kuhn length of the protein polymer from 1.18 nm to 0.58 nm, which we have explained by modifying the freely jointed chain model. Finally, we correlated polymer chain physics to the folding dynamics, and thus provided an analytical framework for understanding compaction-induced folding mechanics across a range of ionic strengths from a single experiment.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

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 redox-controlled chaperone activity of DsbA, on both cysteine-containing and cysteine-free substrates, using magnetic tweezers-based single molecule force spectroscopy that enables independent measurements of oxidoreductase activity and chaperone behavior. Interestingly we found that 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 the 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 found that the 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%.