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ABSTRACT The reversible unfolding and refolding of proteins is a regulatory mechanism of tissue elasticity and signalling used by cells to sense and adapt to extracellular and intracellular

mechanical forces. However, most of these proteins exhibit low mechanical stability, posing technical challenges to the characterization of their conformational dynamics under force. Here,

we detail step-by-step instructions for conducting single-protein nanomechanical experiments using ultra-stable magnetic tweezers, which enable the measurement of the equilibrium

conformational dynamics of single proteins under physiologically relevant low forces applied over biologically relevant timescales. We report the basic principles determining the functioning

of the magnetic tweezer instrument, review the protein design strategy and the fluid chamber preparation and detail the procedure to acquire and analyze the unfolding and refolding

trajectories of individual proteins under force. This technique adds to the toolbox of single-molecule nanomechanical techniques and will be of particular interest to those interested in

proteins involved in mechanosensing and mechanotransduction. The procedure takes 4 d to complete, plus an additional 6 d for protein cloning and production, requiring basic expertise in

molecular biology, surface chemistry and data analysis. KEY POINTS * Ultra-stable magnetic tweezers are used for measuring the conformational dynamics of individual proteins at

physiologically relevant low forces and over long timescales. * Magnetic fields are created by using either permanent magnets or a tape head, which generates precisely calibrated forces for

pulling single proteins tethered between a superparamagnetic bead and a functionalized glass substrate. Access through your institution Buy or subscribe This is a preview of subscription

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* Learn about institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS THE ROLE OF SINGLE-PROTEIN ELASTICITY IN MECHANOBIOLOGY Article 24

October 2022 OPTICAL TWEEZERS IN SINGLE-MOLECULE BIOPHYSICS Article 25 March 2021 MAGNETIC TWEEZERS TO CAPTURE THE FAST-FOLDING Λ6-85 IN SLOW MOTION Article Open access 07 January 2025 DATA

AVAILABILITY Example data from Figs. 7 and 10 can be found as Supplementary Data. Modified pFN18a plasmids from Fig. 5 are available in Addgene (pFN18A-HaloTag-Biotin: Addgene plasmid

#206039; pFN18A-HaloTag-SpyCatcher Addgene plasmid #206041). Other data that support the plots within this paper are available from the corresponding author upon reasonable request. CODE

AVAILABILITY Scripts for the fluctuation analysis are included in the Supplementary Data. The data acquisition code can be accessed at https://doi.org/10.5281/zenodo.8092186. REFERENCES *

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ACKNOWLEDGEMENTS We are deeply grateful to J. Fernandez and C. Badilla (Columbia University) for their pioneering work on technique development and protein engineering and for their legacy

in the field. We thank S. Board, J. Walker and P. Paracuellos for help in protein expression and purification. This work was supported in part by the Francis Crick Institute, which receives

its core funding from Cancer Research U.K. (CC0102), the U.K. Medical Research Council (CC0102) and the Wellcome Trust (CC0102). R.T.-R. is the recipient of a King’s Prize Fellowship. This

work was supported by the European Commission (Mechanocontrol, Grant Agreement 731957), BBSRC sLoLa (BB/V003518/1), Leverhulme Trust Research Leadership Award RL 2016-015, Wellcome Trust

Investigator Award 212218/Z/18/Z and Royal Society Wolfson Fellowship RSWF/R3/183006 to S.G.-M. AUTHOR INFORMATION Author notes * These authors contributed equally: Rafael Tapia-Rojo, Marc

Mora. AUTHORS AND AFFILIATIONS * Single Molecule Mechanobiology Laboratory, The Francis Crick Institute, London, UK Rafael Tapia-Rojo, Marc Mora & Sergi Garcia-Manyes * Department of

Physics, Randall Centre for Cell and Molecular Biophysics, Centre for the Physical Science of Life and London Centre for Nanotechnology, King’s College London, London, UK Rafael Tapia-Rojo, 

Marc Mora & Sergi Garcia-Manyes Authors * Rafael Tapia-Rojo View author publications You can also search for this author inPubMed Google Scholar * Marc Mora View author publications You

can also search for this author inPubMed Google Scholar * Sergi Garcia-Manyes View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS R.T.-R, M.M.

and S.G.-M wrote the paper. CORRESPONDING AUTHORS Correspondence to Rafael Tapia-Rojo, Marc Mora or Sergi Garcia-Manyes. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no

competing interests. PEER REVIEW PEER REVIEW INFORMATION _Nature Protocols_ thanks Tony Huang and the other, anonymous, reviewer(s) for their contribution to the peer review process of this

work. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. RELATED LINKS KEY

REFERENCE USING THIS PROTOCOL Tapia-Rojo, R. et al. _Nat. Phys_. 19, 52–60 (2023): https://doi.org/10.1038/s41567-022-01808-4 EXTENDED DATA EXTENDED DATA FIG. 1 CALCULATING THE STIFFNESS OF

THE MAGNETIC TRAP. Stiffness of the magnetic trap created by the N52 magnets (voice-coil configuration) (A) and magnetic tape head (B). The magnetic trap stiffnesses can be simply calculated

as _dF/dz_, where _z_ is the distance between the gap (magnets or tape head) and the magnetic bead. Because of the nonlinearity of _F_(_z_), the stiffness changes over the control parameter

(magnet position or electric current), but in the operating regime of the trap this results in a very soft trap (~10−4 pN/nm), resulting in effective force clamp conditions (no appreciable

change in force over the range in which the bead moves). EXTENDED DATA FIG. 2 CALIBRATION OF THE TWEEZERS. Calibration of the voice coil-based (A) or tape head–based (B) magnetic tweezers

using the worm-like chain model for polymer elasticity (left) and comparison of the calibration using the worm-like chain (WLC) and freely jointed chain (FJC) (right). The FJC gives a lower

contour length (_ΔL_c = 16.3 nm) compared to the WLC (_ΔL_c = 18.6 nm). All error bars are s.d. EXTENDED DATA FIG. 3 TAPE HEAD AND MAGNETS. The magnetic tape head and voice-coil-mounted

permanent magnets with a magnification of the gap region. SUPPLEMENTARY INFORMATION REPORTING SUMMARY SUPPLEMENTARY DATA 1 Raw traces from talin R3IVVI pulled at 1 pN/s and protein L pulled

at 5 and 10 pN/s SUPPLEMENTARY DATA 2 Raw trace and fluctuation analysis of talin R3IVVI pulled at 8.5 pN SUPPLEMENTARY VIDEOS 1–3 1, how to pull on a protein by using single-molecule

magnetic tweezers; 2, how to assemble the fluid chambers; 3, how to calibrate the distance between the bottom glass cover slide and the magnets RIGHTS AND PERMISSIONS Springer Nature or its

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Tapia-Rojo, R., Mora, M. & Garcia-Manyes, S. Single-molecule magnetic tweezers to probe the equilibrium dynamics of individual proteins at physiologically relevant forces and timescales.

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