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Single-molecule magnetic tweezers to probe the equilibrium dynamics of individual proteins at physiologically relevant forces and timescales

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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.

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Fig. 1: Schematics of the workflow to study the nanomechanics of an individual protein by using single-molecule magnetic tweezers.
Fig. 2: Overview of magnetic tweezers force spectroscopy.
Fig. 3: Image analysis algorithm.
Fig. 4: Step-size-based calibration of magnetic tweezers.
Fig. 5: Protein construct designs and click-chemistry strategies.
Fig. 6: Fluid chamber configuration and assembly.
Fig. 7: Characterization of the mechanical stability of single proteins by using the force-ramp mode.
Fig. 8: Characterization of single-protein folding dynamics by using the constant force mode.
Fig. 9: Measuring long-equilibrium dynamics of individual proteins under force.
Fig. 10: Fluctuation analysis method to fingerprint protein conformations.

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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.

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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.

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Author notes

  1. These authors contributed equally: Rafael Tapia-Rojo, Marc Mora.

Authors and Affiliations

  1. Single Molecule Mechanobiology Laboratory, The Francis Crick Institute, London, UK

    Rafael Tapia-Rojo, Marc Mora & Sergi Garcia-Manyes

  2. 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

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R.T.-R, M.M. and S.G.-M wrote the paper.

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Correspondence to Rafael Tapia-Rojo, Marc Mora or Sergi Garcia-Manyes.

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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 (ΔLc = 16.3 nm) compared to the WLC (ΔLc = 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

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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. Nat Protoc (2024). https://doi.org/10.1038/s41596-024-00965-5

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