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

Nature Protocols volume 19, pages 1779–1806 (2024)Cite this article

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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.

References

  1. Swift, J. et al. Nuclear lamin-A scales with tissue stiffness and enhances matrix-directed differentiation. Science 341, 1240104 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  2. De Belly, H., Paluch, E. K. & Chalut, K. J. Interplay between mechanics and signalling in regulating cell fate. Nat. Rev. Mol. Cell Biol. 23, 465–480 (2022).

    Article  PubMed  Google Scholar 

  3. Iskratsch, T., Wolfenson, H. & Sheetz, M. P. Appreciating force and shape—the rise of mechanotransduction in cell biology. Nat. Rev. Mol. Cell Biol. 15, 825–833 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Paul, C. D., Mistriotis, P. & Konstantopoulos, K. Cancer cell motility: lessons from migration in confined spaces. Nat. Rev. Cancer 17, 131–140 (2017).

    Article  CAS  PubMed  Google Scholar 

  5. Dumortier, J. G. et al. Hydraulic fracturing and active coarsening position the lumen of the mouse blastocyst. Science 365, 465–468 (2019).

    Article  CAS  PubMed  Google Scholar 

  6. Mongera, A. et al. A fluid-to-solid jamming transition underlies vertebrate body axis elongation. Nature 561, 401–405 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Pesce, M. et al. Cardiac fibroblasts and mechanosensation in heart development, health and disease. Nat. Rev. Cardiol. 20, 309–324 (2022).

    Article  PubMed  Google Scholar 

  8. Kefauver, J. M., Ward, A. B. & Patapoutian, A. Discoveries in structure and physiology of mechanically activated ion channels. Nature 587, 567–576 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Huse, M. Mechanical forces in the immune system. Nat. Rev. Immunol. 17, 679–690 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Romani, P., Valcarcel-Jimenez, L., Frezza, C. & Dupont, S. Crosstalk between mechanotransduction and metabolism. Nat. Rev. Mol. Cell Biol. 22, 22–38 (2021).

    Article  CAS  PubMed  Google Scholar 

  11. Romani, P. et al. Mitochondrial fission links ECM mechanotransduction to metabolic redox homeostasis and metastatic chemotherapy resistance. Nat. Cell Biol. 24, 168–180 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Jaalouk, D. E. & Lammerding, J. Mechanotransduction gone awry. Nat. Rev. Mol. Cell Biol. 10, 63–73 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Chemla, Y. R. et al. Mechanism of force generation of a viral DNA packaging motor. Cell 122, 683–692 (2005).

    Article  CAS  PubMed  Google Scholar 

  14. Ranade, S. S., Syeda, R. & Patapoutian, A. Mechanically activated ion channels. Neuron 87, 1162–1179 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Neuman, K. C. & Nagy, A. Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy. Nat. Methods 5, 491–505 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Krieg, M. et al. Atomic force microscopy-based mechanobiology. Nat. Rev. Phys. 1, 41–57 (2018).

    Article  Google Scholar 

  17. Bustamante, C. J., Chemla, Y. R., Liu, S. & Wang, M. D. Optical tweezers in single-molecule biophysics. Nat. Rev. Methods Prim. 1, 25 (2021).

    Article  CAS  Google Scholar 

  18. Popa, I., Kosuri, P., Alegre-Cebollada, J., Garcia-Manyes, S. & Fernandez, J. M. Force dependency of biochemical reactions measured by single-molecule force-clamp spectroscopy. Nat. Protoc. 8, 1261–1276 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Li, H. et al. Reverse engineering of the giant muscle protein titin. Nature 418, 998–1002 (2002).

    Article  CAS  PubMed  Google Scholar 

  20. Echelman, D. J., Lee, A. Q. & Fernandez, J. M. Mechanical forces regulate the reactivity of a thioester bond in a bacterial adhesin. J. Biol. Chem. 292, 8988–8997 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Milles, L. F., Schulten, K., Gaub, H. E. & Bernardi, R. C. Molecular mechanism of extreme mechanostability in a pathogen adhesin. Science 359, 1527–1533 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Mora, M., Stannard, A. & Garcia-Manyes, S. The nanomechanics of individual proteins. Chem. Soc. Rev. 49, 6816–6832 (2020).

    Article  CAS  PubMed  Google Scholar 

  23. Dudko, O. K., Hummer, G. & Szabo, A. Theory, analysis, and interpretation of single-molecule force spectroscopy experiments. Proc. Natl Acad. Sci. USA 105, 15755–15760 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Cecconi, C., Shank, E. A., Bustamante, C. & Marqusee, S. Direct observation of the three-state folding of a single protein molecule. Science 309, 2057–2060 (2005).

    Article  CAS  PubMed  Google Scholar 

  25. Stigler, J., Ziegler, F., Gieseke, A., Gebhardt, J. C. & Rief, M. The complex folding network of single calmodulin molecules. Science 334, 512–516 (2011).

    Article  CAS  PubMed  Google Scholar 

  26. Neupane, K., Manuel, A. P. & Woodside, M. T. Protein folding trajectories can be described quantitatively by one-dimensional diffusion over measured energy landscapes. Nat. Phys. 12, 700–703 (2016).

    Article  CAS  Google Scholar 

  27. Woodside, M. T. & Block, S. M. Reconstructing folding energy landscapes by single-molecule force spectroscopy. Annu. Rev. Biophys. 43, 19–39 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Kaiser, C. M., Goldman, D. H., Chodera, J. D., Tinoco, I. Jr. & Bustamante, C. The ribosome modulates nascent protein folding. Science 334, 1723–1727 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Lipfert, J., Kerssemakers, J. W., Jager, T. & Dekker, N. H. Magnetic torque tweezers: measuring torsional stiffness in DNA and RecA-DNA filaments. Nat. Methods 7, 977–980 (2010).

    Article  CAS  PubMed  Google Scholar 

  30. Ding, F. et al. Single-molecule mechanical identification and sequencing. Nat. Methods 9, 367–372 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Hodeib, S. et al. Single molecule studies of helicases with magnetic tweezers. Methods 105, 3–15 (2016).

    Article  CAS  PubMed  Google Scholar 

  32. Lionnet, T. et al. Magnetic trap construction. Cold Spring Harb. Protoc. 2012, 133–138 (2012).

    Article  PubMed  Google Scholar 

  33. Rivas-Pardo, J. A. et al. Work done by titin protein folding assists muscle contraction. Cell Rep. 14, 1339–1347 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Chen, H. et al. Dynamics of equilibrium folding and unfolding transitions of titin immunoglobulin domain under constant forces. J. Am. Chem. Soc. 137, 3540–3546 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Bauer, M. S. et al. A tethered ligand assay to probe SARS-CoV-2:ACE2 interactions. Proc. Natl Acad. Sci. USA 119, e2114397119 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Yao, M. et al. The mechanical response of talin. Nat. Commun. 7, 11966 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  37. Popa, I. et al. A HaloTag anchored ruler for week-long studies of protein dynamics. J. Am. Chem. Soc. 138, 10546–10553 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Lof, A. et al. Multiplexed protein force spectroscopy reveals equilibrium protein folding dynamics and the low-force response of von Willebrand factor. Proc. Natl Acad. Sci. USA 116, 18798–18807 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  39. Zhao, X., Zeng, X., Lu, C. & Yan, J. Studying the mechanical responses of proteins using magnetic tweezers. Nanotechnology 28, 414002 (2017).

    Article  PubMed  Google Scholar 

  40. Choi, H. K. et al. Watching helical membrane proteins fold reveals a common N-to-C-terminal folding pathway. Science 366, 1150–1156 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Choi, H. K. et al. Evolutionary balance between foldability and functionality of a glucose transporter. Nat. Chem. Biol. 18, 713–723 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Choi, H. K., Kim, H. G., Shon, M. J. & Yoon, T. Y. High-resolution single-molecule magnetic tweezers. Annu. Rev. Biochem. 91, 33–59 (2022).

    Article  CAS  PubMed  Google Scholar 

  43. Smith, S. B., Finzi, L. & Bustamante, C. Direct mechanical measurements of the elasticity of single DNA molecules by using magnetic beads. Science 258, 1122–1126 (1992).

    Article  CAS  PubMed  Google Scholar 

  44. Strick, T. R., Allemand, J. F., Bensimon, D., Bensimon, A. & Croquette, V. The elasticity of a single supercoiled DNA molecule. Science 271, 1835–1837 (1996).

    Article  CAS  PubMed  Google Scholar 

  45. Gosse, C. & Croquette, V. Magnetic tweezers: micromanipulation and force measurement at the molecular level. Biophys. J. 82, 3314–3329 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Maier, B., Bensimon, D. & Croquette, V. Replication by a single DNA polymerase of a stretched single-stranded DNA. Proc. Natl Acad. Sci. USA 97, 12002–12007 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Dekker, N. H. et al. The mechanism of type IA topoisomerases. Proc. Natl Acad. Sci. USA 99, 12126–12131 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Crut, A., Koster, D. A., Seidel, R., Wiggins, C. H. & Dekker, N. H. Fast dynamics of supercoiled DNA revealed by single-molecule experiments. Proc. Natl Acad. Sci. USA 104, 11957–11962 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. England, C. G., Luo, H. & Cai, W. HaloTag technology: a versatile platform for biomedical applications. Bioconjug. Chem. 26, 975–986 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Zakeri, B. et al. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc. Natl Acad. Sci. USA 109, E690–E697 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Tapia-Rojo, R., Eckels, E. C. & Fernandez, J. M. Ephemeral states in protein folding under force captured with a magnetic tweezers design. Proc. Natl Acad. Sci. USA 116, 7873–7878 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Stannard, A. et al. Molecular fluctuations as a ruler of force-induced protein conformations. Nano Lett. 21, 2953–2961 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Tapia-Rojo, R., Alonso-Caballero, A. & Fernandez, J. M. Direct observation of a coil-to-helix contraction triggered by vinculin binding to talin. Sci. Adv. 6, eaaz4707 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Franz, F. et al. Allosteric activation of vinculin by talin. Nat. Commun. 14, 4311 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Tapia-Rojo, R. et al. Enhanced statistical sampling reveals microscopic complexity in the talin mechanosensor folding energy landscape. Nat. Phys. 19, 52–60 (2023).

    Article  CAS  PubMed  Google Scholar 

  56. Austen, K. et al. Extracellular rigidity sensing by talin isoform-specific mechanical linkages. Nat. Cell Biol. 17, 1597–1606 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Riveline, D. et al. Focal contacts as mechanosensors: externally applied local mechanical force induces growth of focal contacts by an mDia1-dependent and ROCK-independent mechanism. J. Cell Biol. 153, 1175–1186 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Grashoff, C. et al. Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics. Nature 466, 263–266 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Garcia-Manyes, S. et al. Single-molecule force spectroscopy predicts a misfolded, domain-swapped conformation in human γD-crystallin protein. J. Biol. Chem. 291, 4226–4235 (2016).

    Article  CAS  PubMed  Google Scholar 

  60. Mora, M. et al. A single-molecule strategy to capture non-native intramolecular and intermolecular protein disulfide bridges. Nano Lett. 22, 3922–3930 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Petrosyan, R., Patra, S., Rezajooei, N., Garen, C. R. & Woodside, M. T. Unfolded and intermediate states of PrP play a key role in the mechanism of action of an antiprion chaperone. Proc. Natl Acad. Sci. USA 118, e2010213118 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Gupta, A. N. et al. Pharmacological chaperone reshapes the energy landscape for folding and aggregation of the prion protein. Nat. Commun. 7, 12058 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Sen Mojumdar, S. et al. Partially native intermediates mediate misfolding of SOD1 in single-molecule folding trajectories. Nat. Commun. 8, 1881 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  64. Yao, M. et al. Force-dependent conformational switch of alpha-catenin controls vinculin binding. Nat. Commun. 5, 4525 (2014).

    Article  CAS  PubMed  Google Scholar 

  65. Dahal, N., Sharma, S., Phan, B., Eis, A. & Popa, I. Mechanical regulation of talin through binding and history-dependent unfolding. Sci. Adv. 8, eabl7719 (2022).

    Article  CAS  PubMed  Google Scholar 

  66. Kemmerich, F. E. et al. Simultaneous single-molecule force and fluorescence sampling of DNA nanostructure conformations using magnetic tweezers. Nano Lett. 16, 381–386 (2016).

    Article  CAS  PubMed  Google Scholar 

  67. Ivanov, I. E. et al. Multimodal measurements of single-molecule dynamics using FluoRBT. Biophys. J. 114, 278–282 (2018).

    Article  CAS  PubMed  Google Scholar 

  68. Tapia-Rojo, R., Alonso-Caballero, A. & Fernandez, J. M. Talin folding as the tuning fork of cellular mechanotransduction. Proc. Natl Acad. Sci. USA 117, 21346–21353 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Alonso-Caballero, A. et al. Protein folding modulates the chemical reactivity of a Gram-positive adhesin. Nat. Chem. 13, 172–181 (2021).

    Article  CAS  PubMed  Google Scholar 

  70. Guo, H. A simple algorithm for fitting a gaussian function. In Streamlining Digital Signal Processing 297–305 (John Wiley & Sons, 2012).

  71. Fonnum, G., Johansson, C., Molteberg, A., Mørup, S. & Aksnes, E. Characterisation of Dynabeads® by magnetization measurements and Mössbauer spectroscopy. J. Magn. Magn. Mater. 293, 41–47 (2005).

    Article  CAS  Google Scholar 

  72. Ostrofet, E., Papini, F. S. & Dulin, D. Correction-free force calibration for magnetic tweezers experiments. Sci. Rep. 8, 15920 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  73. Buschow, K. H. J., Long, G. J. & Grandjean, F. High Density Digital Recording (Springer, 1993).

  74. Liu, R., Garcia-Manyes, S., Sarkar, A., Badilla, C. L. & Fernandez, J. M. Mechanical characterization of protein L in the low-force regime by electromagnetic tweezers/evanescent nanometry. Biophys. J. 96, 3810–3821 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Valle-Orero, J. et al. Proteins breaking bad: a free energy perspective. J. Phys. Chem. Lett. 8, 3642–3647 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Alegre-Cebollada, J., Badilla, C. L. & Fernandez, J. M. Isopeptide bonds block the mechanical extension of pili in pathogenic Streptococcus pyogenes. J. Biol. Chem. 285, 11235–11242 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Schlierf, M., Li, H. & Fernandez, J. M. The unfolding kinetics of ubiquitin captured with single-molecule force-clamp techniques. Proc. Natl Acad. Sci. USA 101, 7299–7304 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Evans, E. & Ritchie, K. Dynamic strength of molecular adhesion bonds. Biophys. J. 72, 1541–1555 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. Zhang, Y., Jiao, J. & Rebane, A. A. Hidden Markov modeling with detailed balance and its application to single protein folding. Biophys. J. 111, 2110–2124 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. McKinney, S. A., Joo, C. & Ha, T. Analysis of single-molecule FRET trajectories using hidden Markov modeling. Biophys. J. 91, 1941–1951 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Dudko, O. K., Hummer, G. & Szabo, A. Intrinsic rates and activation free energies from single-molecule pulling experiments. Phys. Rev. Lett. 96, 108101 (2006).

    Article  PubMed  Google Scholar 

  82. Bullerjahn, J. T., Sturm, S. & Kroy, K. Theory of rapid force spectroscopy. Nat. Commun. 5, 4463 (2014).

    Article  CAS  PubMed  Google Scholar 

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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 19, 1779–1806 (2024). https://doi.org/10.1038/s41596-024-00965-5

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