The Power of Force: Insights into the Protein Folding Process Using Single-Molecule Force Spectroscopy

Schönfelder, Jörg; Sancho, David De; Pérez-Jiménez, Raúl

doi:10.1016/j.jmb.2016.09.006

Cited by 29 publications

(23 citation statements)

References 139 publications

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“…Single molecule force spectroscopy (smFS) experiments, either using the AFM or optical tweezers (OT), are now an essential part of the toolbox for the study of the conformational dynamics of biomolecules 1–2 . While in conventional experiments one measures the behavior of an ensemble of molecules of the protein or nucleic acid of interest, smFS looks at a single copy of the molecule, which is manipulated by perturbing a well-defined progress variable for the biomolecular process of interest (e.g.…”

Section: Introductionmentioning

confidence: 99%

Instrumental Effects in the Dynamics of an Ultrafast Folding Protein under Mechanical Force

et al. 2018

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The analysis and interpretation of single molecule force spectroscopy (smFS) experiments is often complicated by hidden effects from the measuring device. Here we investigate these effects in our recent smFS experiments on the ultrafast folding protein gpW, which has been previously shown to fold without crossing a free energy barrier in the absence of force (i.e., downhill folding). Using atomic force microscopy (AFM) smFS experiments, we found that a very small force of ∼5 pN brings gpW near its unfolding midpoint and results in two-state (un)folding patterns that indicate the emergence of a force-induced free energy barrier. The change in the folding regime is concomitant with a 30,000-fold slowdown of the folding and unfolding times, from a few microseconds that it takes gpW to (un)fold at the midpoint temperature to seconds in the AFM. These results are puzzling because the barrier induced by force in the folding free energy landscape of gpW is far too small to account for such a difference in time scales. Here we use recently developed theoretical methods to resolve the origin of the strikingly slow dynamics of gpW under mechanical force. We find that, while the AFM experiments correctly capture the equilibrium distance distribution, the measured dynamics are entirely controlled by the response of the cantilever and polyprotein linker, which is much slower than the protein conformational dynamics. This interpretation is likely applicable to the folding of other small biomolecules in smFS experiments, and becomes particularly important in the case of systems with fast folding dynamics and small free energy barriers, and for instruments with slow response times.

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Section: Introductionmentioning

confidence: 99%

Instrumental Effects in the Dynamics of an Ultrafast Folding Protein under Mechanical Force

et al. 2018

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“…The purpose of this focused review is to highlight recent advances in AFM-based SMFS methodology that address the existing limitations and improve aspects such as sample throughput, sensitivity, reliability, and general robustness of the measurement. There are several recent reviews on related topics that overlap with the current review (Chen et al, 2015;Hughes and Dougan, 2016;Schönfelder et al, 2016aSchönfelder et al, , 2018Johnson and Thomas, 2018;Li and Zheng, 2018;Nathwani et al, 2018), and we regret that we were not able to include all the relevant work. We have organized the review into three sections.…”

Section: Introductionmentioning

confidence: 99%

Next Generation Methods for Single-Molecule Force Spectroscopy on Polyproteins and Receptor-Ligand Complexes

Yang

Liu

et al. 2020

Front. Mol. Biosci.

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Single-molecule force spectroscopy with the atomic force microscope provides molecular level insights into protein function, allowing researchers to reconstruct energy landscapes and understand functional mechanisms in biology. With steadily advancing methods, this technique has greatly accelerated our understanding of force transduction, mechanical deformation, and mechanostability within single-and multi-domain polyproteins, and receptor-ligand complexes. In this focused review, we summarize the state of the art in terms of methodology and highlight recent methodological improvements for AFM-SMFS experiments, including developments in surface chemistry, considerations for protein engineering, as well as theory and algorithms for data analysis. We hope that by condensing and disseminating these methods, they can assist the community in improving data yield, reliability, and throughput and thereby enhance the information that researchers can extract from such experiments. These leading edge methods for AFM-SMFS will serve as a groundwork for researchers cognizant of its current limitations who seek to improve the technique in the future for in-depth studies of molecular biomechanics.

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“…26,27 Mechanical stability describes how much tension a folded domain can withstand prior to unfolding, or how much force is required to dissociate a receptor-ligand complex. Singlemolecule force spectroscopy (SMFS) [28][29][30][31] with the atomic force microscope (AFM) can be used to stretch single protein molecules, quantify intermediate folding states 32,33 elucidate [un]folding energy landscapes while accounting for differences in loading geometry [34][35][36][37][38] or the presence of dual modes of ligand recognition 39,40 . The goal of this work was therefore to investigate the role of protein fluorination on non-equilibrium mechanostability, specifically investigating any discrepancies in trends between equilibrium thermodynamic stability and non-equilibrium mechanostability.…”

Section: Main Text Introductionmentioning

confidence: 99%

Influence of Fluorination on Single-Molecule Unfolding and Rupture Pathways of a Mechanostable Protein Adhesion Complex

Yang

Liu

et al. 2020

Preprint

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AbstractFluorination of proteins by cotranslational incorporation of non-canonical amino acids is a valuable tool for enhancing biophysical stability. Despite many prior studies investigating the effects of fluorination on equilibrium stability, its influence on non-equilibrium mechanical stability remains unknown. Here, we used single-molecule force spectroscopy (SMFS) with the atomic force microscope (AFM) to investigate the influence of fluorination on unfolding and unbinding pathways of a mechanically ultrastable bacterial adhesion complex. We assembled modular polyproteins comprising the tandem dyad XModule-Dockerin (XMod-Doc) bound to a globular Cohesin (Coh) domain. By applying tension across the binding interface, and quantifying single-molecule unfolding and rupture events, we mapped the energy landscapes governing the unfolding and unbinding reactions. We then used sense codon suppression to substitute trifluoroleucine (TFL) in place of canonical leucine (LEU) globally in XMod-Doc, or selectively within the Doc subdomain of a mutant XMod-Doc. Although TFL substitution thermally destabilized XMod-Doc, it had little effect on XMod-Doc:Coh binding affinity at equilibrium. When we mechanically dissociated global TFL-substituted XMod-Doc from Coh, we observed the emergence of a new unbinding pathway with a lower energy barrier. Counterintuitively, when fluorination was restricted to Doc, we observed mechano-stabilization of the non-fluorinated neighboring XMod domain. These results suggest that intramolecular deformation networks can be modulated by fluorination, and further highlight significant differences between equilibrium thermostability, where all constructs were destabilized, and non-equilibrium mechanostability, where XMod was strengthened. Future work is poised to investigate the influence of non-natural amino acids on mechanically-accelerated protein unfolding and unbinding reaction pathways.

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The Power of Force: Insights into the Protein Folding Process Using Single-Molecule Force Spectroscopy

Cited by 29 publications

References 139 publications

Instrumental Effects in the Dynamics of an Ultrafast Folding Protein under Mechanical Force

Instrumental Effects in the Dynamics of an Ultrafast Folding Protein under Mechanical Force

Next Generation Methods for Single-Molecule Force Spectroscopy on Polyproteins and Receptor-Ligand Complexes

Influence of Fluorination on Single-Molecule Unfolding and Rupture Pathways of a Mechanostable Protein Adhesion Complex

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