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

Chakraborty, Soham; Chaudhuri, Deep; Banerjee, Souradeep; Bhatt, Madhu; Haldar, Shubhasis

doi:10.1038/s42003-022-03258-3

Cited by 15 publications

(28 citation statements)

References 80 publications

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“…The changes in length between the centers of two histogram peaks denote the step size. The force calibration has been shown in detail in our previous paper . We have discussed the image processing in the Supporting Information (Figures S1 and S2).…”

Section: Experimental (Materials and Methods)mentioning

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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Section: Experimental (Materials and Methods)mentioning

confidence: 99%

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

et al. 2022

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“…Forces were applied through the generation of magnetic field by neodymium magnets, which are attached to the linear voice coil (Equipment Solutions) the force can be controlled by changing the position of the magnets. Detail information on force calibration, bead tracking and image processing were discussed previously by Chakraborty et al 41 . Experiments were performed in flow chamber with 1-10 nM protein in 1X PBS buffer (pH 7.2).…”

Section: Methodsmentioning

confidence: 99%

Talin-drug interaction reveals a key molecular determinant for biphasic mechanical effect: studied under single-molecule resolution

Chakraborty

Bhatt

Chowdhury

et al. 2022

Preprint

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Talin as an adhesion protein, exhibits a strong force-dependent structure-function dynamics. Being a mechanosensitive focal adhesion (FA) protein, talin might interact to several FA targeting drugs; however, the molecular mechanism of talin-drug interactions remains elusive. Here we combined magnetic tweezers and molecular dynamics (MD) simulation to explore mechanical stability of talin with three drugs based on their talin specificity. Interestingly, our study revealed that talin displays a bimodal force distribution with a low and high unfolding force population. We observed that talin nonspecific drugs (tamoxifen and letrozole) display biphasic effect: increase talin mechanical stability upto optimum concentration, followed by a decrease in stability with further concentration increase. By contrast, talin-specific cyanidin 3-O-glucoside promotes a steady increase to talin mechanical stability with its concentration. We reconciled our observation from the simulation study: tamoxifen enters into talin hydrophobic core, eventually destabilizing the protein; whereas cyanidin 3-O-glucoside stabilizes the protein core by maintaining the inter-helix distance. Finally, we observed a strong correlation among hydrophobicity and cavity analysis, illustrating a detailed mechanistic analysis of drug effect on the mechanosensitive protein. Overall this study presents a novel perspective for drug designing against mechanosensitive proteins and studying off-target effects of already known drugs.

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“…The N terminus of the polyprotein is tethered to a glass surface via HaloTag covalent chemistry, while the C terminus is attached with streptavidin-coated paramagnetic bead. Force is applied by introducing a pair of permanent magnets that can exert a magnetic field vertically towards the tethered protein 13,14,17 (Fig. 1A).…”

Section: Folding Dynamics Of Protein L Measured By Single-molecule Ma...mentioning

confidence: 99%

“…Here, we have investigated the TMAO effect on protein L as a model substrate, which has previously been used in force spectroscopic studies [14][15][16][17] . Since protein L is not biologically relevant to function under mechanical tension, we selected another substrate talin, which is well-known to work under physiological force and extensively used in force spectroscopy studies 13,[18][19][20] . Interestingly, these two substrates are structurally different: protein L is predominantly a β-sheet containing substrate, whereas talin is an α-helical protein.…”

Section: Introductionmentioning

confidence: 99%

“…To address this question, we have performed single-molecule magnetic tweezers spectroscopy, which employs both the force-ramp and force-clamp methodology 13,14 . Using the force-ramp methodology, the applied force can be increased or decreased at a constant loading rate, detecting unfolding and refolding events at different forces on a single molecule; while the force-clamp methodology at a constant applied force allow us to measure the TMAO effect on both the folding dynamics and kinetics under equilibrium condition.…”

Section: Introductionmentioning

confidence: 99%

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Trimethylamine N-oxide (TMAO) enhances substrate mechanical stability probed by single molecule magnetic tweezers

Chaudhuri

Chowdhury

Chakraborty

et al. 2022

Preprint

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Trimethylamine N-oxide (TMAO) is a well-known osmolyte to stabilize the folded proteins through a variety of mechanisms. Since mechanical strength of proteins is a critical determinant of its stabilization, TMAO might play a relevant role by favoring its folding dynamics or by enhancing its mechanical stability. To address this question, we have performed the single-molecule magnetic tweezers experiment to explore the TMAO effect on two structurally distinct substrates-protein L and talin. We observed that TMAO increases the mechanical stability of these proteins through increasing their unfolding force. Additionally, we are able to demonstrate that TMAO retards the unfolding kinetics, while accelerating the refolding kinetics under force; which eventually tilts the energy landscape towards the folded state. Interestingly, this TMAO-enhanced protein folding generates mechanical work output upto ∼67 zJ, allowing the protein folding under higher force regime. Overall this TMAO-enhanced mechanical stability provides a significant implication to folding-induced structural stability of proteins.

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

Cited by 15 publications

References 80 publications

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

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

Talin-drug interaction reveals a key molecular determinant for biphasic mechanical effect: studied under single-molecule resolution

Trimethylamine N-oxide (TMAO) enhances substrate mechanical stability probed by single molecule magnetic tweezers

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