DsbA is a redox-switchable mechanical chaperone

Eckels, Edward C.; Chaudhuri, Deep; Chakraborty, Soham; Echelman, Daniel J.; Haldar, Shubhasis

doi:10.1039/d1sc03048e

Cited by 11 publications

(17 citation statements)

References 66 publications

(87 reference statements)

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“…2c ). Remarkably, in the presence of 60 µM oxidized DsbA, a known force-dependent foldase chaperone 24 , the mechanical stability of talin increased to 14.3 ± 0.5 pN (Fig. 2d ).…”

Section: Resultsmentioning

confidence: 92%

“…By contrast, tunnel-associated chaperones possess the ability to increase the mechanical stability of their substrates. Such as oxidoreductase DsbA, a well-known model mechanical foldase, has the ability to increase the folding rate and mechanical stability of R3 24 , 77 , 78 . Hence, our results demonstrate that chaperones could modulate the mechanical stability of the FA protein talin, which is characterized by the change in the unfolding free energy barrier.…”

Section: Discussionmentioning

confidence: 99%

“…The protein was further purified by size exclusion chromatography using Superdex-200 increase 10/300 GL gel filtration column in presence of 150 mM NaCl (Supplementary Fig. 16 ) 24 . We purchased Hsp70 and Hsp40 chaperones from ORIGENE Technologies.…”

Section: Methodsmentioning

confidence: 99%

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Direct observation of chaperone-modulated talin mechanics with single-molecule resolution

et al. 2022

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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.

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“…2c ). Remarkably, in the presence of 60 µM oxidized DsbA, a known force-dependent foldase chaperone 24 , the mechanical stability of talin increased to 14.3 ± 0.5 pN (Fig. 2d ).…”

Section: Resultsmentioning

confidence: 92%

Section: Discussionmentioning

confidence: 99%

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Direct observation of chaperone-modulated talin mechanics with single-molecule resolution

et al. 2022

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“…The periplasmic oxidoreductase enzyme DsbA has a well-characterized function to transfer the disulfide bond to substrate proteins by reducing its catalytic CXXC motif and helps in the folding of cysteine-containing proteins. − Interestingly, few studies revealed the role of DsbA in transporting cysteine-free proteins through translocon pores, suggesting its plausible chaperone activity independent of oxidoreductase activity. − Therefore, we explore the mechanical chaperone activity of DsbA by using our substrate protein L, a cysteine-free globular protein. Unlike DnaJ or SecB, the refolding kinetics is greatly increased in the presence of DsbA, while a slight change was observed in unfolding kinetics (Figure D,E).…”

Section: Resultsmentioning

confidence: 99%

Direct Observation of the Mechanical Role of Bacterial Chaperones in Protein Folding

et al. 2022

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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.

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“…6). Since protein folding at the edge of any translocon pore act as a source of force generation (10,19), these chaperones might influence the translocation of the substrates (which was not certified by peer review) is the author/funder. All rights reserved.…”

Section: Discussionmentioning

confidence: 99%

Force-regulated chaperone activity of BiP/ERdj3 is opposite to their homologs DnaK/DnaJ: explained by strain energy

Haldar

Banerjee

Chowdhury

et al. 2023

Preprint

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Polypeptide chains experiences mechanical tension while translocating through cellular tunnel. In this scenario, interaction of tunnel-associated chaperones with the emerging polypeptide occurs under force; however, this force-regulated chaperone behaviour is not fully understood. We studied the mechanical chaperone activity of two tunnel-associated chaperones BiP and ERdj3 both in the absence and presence of force; and compared to their respective cytoplasmic homologs DnaK and DnaJ. We found that BiP/ERdj3 shows strong foldase activity under force; whereas their cytoplasmic homolog DnaK/DnaJ behave as holdase. Importantly, these tunnel-associated chaperones (BiP/ERdj3) revert to holdase in the absence of force, suggesting that mechanical chaperone activity differs depending on the presence or absence of force. This tunnel-associated chaperone-driven folding event generates additional mechanical energy of up to 54 zJ that could help protein translocation. The mechanical-chaperone behaviour can be explained by strain theory: chaperones with higher intrinsic deformability function as mechanical foldase (BiP, ERdj3), while chaperones with lower intrinsic deformability act as holdase (DnaK and DnaJ). Our study thus unveils the underlying mechanism of mechanically regulated chaperoning activity and provides a novel mechanism of co-translocational protein folding.

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DsbA is a redox-switchable mechanical chaperone

Abstract: 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...

Cited by 11 publications

References 66 publications

Direct observation of chaperone-modulated talin mechanics with single-molecule resolution

Direct observation of chaperone-modulated talin mechanics with single-molecule resolution

Direct Observation of the Mechanical Role of Bacterial Chaperones in Protein Folding

Force-regulated chaperone activity of BiP/ERdj3 is opposite to their homologs DnaK/DnaJ: explained by strain energy

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