Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Force-activated reactivity switch in a bimolecular chemical reaction

Abstract

The effect of mechanical force on the free-energy surface that governs a chemical reaction is largely unknown. The combination of protein engineering with single-molecule force-clamp spectroscopy allows us to study the influence of mechanical force on the rate at which a protein disulfide bond is reduced by nucleophiles in a bimolecular substitution reaction (SN2). We found that cleavage of a protein disulfide bond by hydroxide anions exhibits an abrupt reactivity ‘switch’ at 500 pN, after which the accelerating effect of force on the rate of an SN2 chemical reaction greatly diminishes. We propose that an abrupt force-induced conformational change of the protein disulfide bond shifts its ground state, drastically changing its reactivity in SN2 chemical reactions. Our experiments directly demonstrate the action of a force-activated switch in the chemical reactivity of a single molecule.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Force-clamp spectroscopy monitors events in the reduction of single disulfide bonds with hydroxide anions in the solution.
Figure 2: The force dependency of the rate of disulfide-bond reduction by hydroxide anions exhibits two distinct reactivity regimes that switch at 500 pN.
Figure 3: The reactivity switch observed at a 500 pN is a general mechanism in force-activated SN2 reactions.
Figure 4: The reactivity switch is independent of the location of the disulfide bond in the protein structure.
Figure 5: Schematics of the two energy scenarios compatible with the experimental results.

Similar content being viewed by others

References

  1. Beyer, M. K. & Clausen-Schaumann, H. Mechanochemistry: The mechanical activation of covalent bonds. Chem. Rev. 105, 2921–2948 (2005).

    Article  CAS  Google Scholar 

  2. Hickenboth, C. R. et al. Biasing reaction pathways with mechanical force. Nature 446, 423–427 (2007).

    Article  CAS  Google Scholar 

  3. Beyer, M. K. Coupling of mechanical and chemical energy: proton affinity as a function of external force. Angew. Chem. Int. Ed. 42, 4913–4915 (2003).

    Article  CAS  Google Scholar 

  4. Grandbois, M., Beyer, M., Rief, M., Clausen-Schaumann, H. & Gaub, H. E. How strong is a covalent bond? Science 283, 1727–1730 (1999).

    Article  CAS  Google Scholar 

  5. Garnier, L., Gauthier-Manuel, B., van der Vegte, E. W., Snijders, J. & Hadziioannou, G. Covalent bond force profile and cleavage in a single polymer chain. J. Chem. Phys. 113, 2497–2503 (2000).

    Article  CAS  Google Scholar 

  6. Schmidt, S. W., Beyer, M. K. & Clausen-Schaumann, H. Dynamic strength of the silicon–carbon bond observed over three decades of force-loading rates. J. Am. Chem. Soc. 130, 3664–3668 (2008).

    Article  CAS  Google Scholar 

  7. Aktah, D. & Frank, I. Breaking bonds by mechanical stress: when do electrons decide for the other side? J. Am. Chem. Soc. 124, 3402–3406 (2002).

    Article  CAS  Google Scholar 

  8. Beyer, M. K. The mechanical strength of a covalent bond calculated by density functional theory. J. Chem. Phys. 112, 7307–7312 (2000).

    Article  CAS  Google Scholar 

  9. Liphardt, J., Onoa, B., Smith, S. B., Tinoco, I. & Bustamante, C. Reversible unfolding of single RNA molecules by mechanical force. Science 292, 733–737 (2001).

    Article  CAS  Google Scholar 

  10. Gore, J. et al. DNA overwinds when stretched. Nature 442, 836–839 (2006).

    Article  CAS  Google Scholar 

  11. Woodside, M. T. et al. Direct measurement of the full, sequence-dependent folding landscape of a nucleic acid. Science 314, 1001–1004 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  13. Rief, M., Gautel, M., Oesterhelt, F., Fernandez, J. M. & Gaub, H. E. Reversible unfolding of individual titin immunoglobulin domains by AFM. Science 276, 1109–1112 (1997).

    Article  CAS  Google Scholar 

  14. Kellermayer, M. S. Z., Smith, S. B., Granzier, H. L. & Bustamante, C. Folding–unfolding transitions in single titin molecules characterized with laser tweezers. Science 276, 1112–1116 (1997).

    Article  CAS  Google Scholar 

  15. 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  Google Scholar 

  16. Marszalek, P. E., Oberhauser, A. F., Pang, Y. P. & Fernandez, J. M. Polysaccharide elasticity governed by chair–boat transitions of the glucopyranose ring. Nature 396, 661–664 (1998).

    Article  CAS  Google Scholar 

  17. Wiita, A. P., Ainavarapu, S. R., Huang, H. H. & Fernandez, J. M. Force-dependent chemical kinetics of disulfide bond reduction observed with single-molecule techniques. Proc. Natl Acad. Sci. USA 103, 7222–7227 (2006).

    Article  CAS  Google Scholar 

  18. Ainavarapu, R. K. et al. Contour length and refolding rate of a small protein controlled by engineered disulfide bonds. Biophys. J. 92, 225–233 (2007).

    Article  CAS  Google Scholar 

  19. Ainavarapu, S. R. K., Wiita, A. P., Dougan, L., Uggerud, E. & Fernandez, J. M. Single-molecule force spectroscopy measurements of bond elongation during a bimolecular reaction. J. Am. Chem. Soc. 130, 6479–6487 (2008).

    Article  CAS  Google Scholar 

  20. Carrion-Vazquez, M. et al. Mechanical and chemical unfolding of a single protein: a comparison. Proc. Natl Acad. Sci. USA 96, 3694–3699 (1999).

    Article  CAS  Google Scholar 

  21. Garcia-Manyes, S., Brujic, J., Badilla, C. L. & Fernandez, J. M. Force-clamp spectroscopy of single-protein monomers reveals the individual unfolding and folding pathways of I27 and ubiquitin. Biophys. J. 93, 2436–2446 (2007).

    Article  CAS  Google Scholar 

  22. Lien-Vien, D., Colthup, N. B., Fateley, W. G. & Grasselli, J. G. The Handbook of Infrared and Raman Characteristic Frequencies of Organic Molecules 231–232 (Academic Press, 1991).

  23. Bickelhaupt, F. M., Baerends, E. J. & Nibbering, N. M. M. The effect of microsolvation on E2 and SN2 reactions: theoretical study of the model system F + C2H5F + nHF. Chem. Eur. J. 2, 196–207 (1996).

    Article  CAS  Google Scholar 

  24. Chandrasekhar, J., Smith, S. F. & Jorgensen, W. L. Theoretical examination of the SN2 reaction involving chloride ion and methyl chloride in the gas phase and aqueous solution. J. Am. Chem. Soc. 107, 154–163 (1985).

    Article  CAS  Google Scholar 

  25. Chandrasekhar, J. & Jorgensen, W. L. Energy profile for a nonconcerted SN2 reaction in solution. J. Am. Chem. Soc. 107, 2974–2975 (1985).

    Article  CAS  Google Scholar 

  26. Bohme, D. K. & Mackay, G. I. Bridging the gap between the gas phase and solution – transition in the kinetics of nucleophilic displacement reactions. J. Am. Chem. Soc. 103, 978–979 (1981).

    Article  CAS  Google Scholar 

  27. Brauman, J. I. Chemistry – Not so simple. Science 319, 168–168 (2008).

    Article  CAS  Google Scholar 

  28. Boyd, D. B. Sequence and shape of molecular-orbitals of disulfide HSSH. J. Phys. Chem. 78, 1554–1563 (1974).

    Article  CAS  Google Scholar 

  29. Boyd, D. B. Mapping electron density in molecules. 14. Nonbonded contacts between lone pairs on divalent sulfurs. J. Phys. Chem. 82, 1407–1416 (1978).

    Article  CAS  Google Scholar 

  30. Vanwart, H. E. & Scheraga, H. A. Stable conformations of aliphatic disulfides – influence of 1,4 interactions involving sulfur atoms. Proc. Natl Acad. Sci. USA 74, 13–17 (1977).

    Article  CAS  Google Scholar 

  31. Jiao, D., Barfield, M., Combariza, J. E. & Hruby, V. J. Ab initio molecular-orbital studies of the rotational barriers and the S-33 and C-13 chemical shieldings for dimethyl disulfide. J. Am. Chem. Soc. 114, 3639–3643 (1992).

    Article  CAS  Google Scholar 

  32. Vanwart, H. E., Shipman, L. L. & Scheraga, H. A. Variation of disulfide bond stretching frequencies with disulfide dihedral angle in dimethyl disulfide. J. Phys. Chem. 78, 1848–1853 (1974).

    Article  CAS  Google Scholar 

  33. Vollhardt, K. P. C. Organic Chemistry (Freeman, 2003).

  34. Uggerud, E. Nucleophilicity – periodic trends and connection to basicity. Chem. Eur. J. 12, 1127–1136 (2006).

    Article  CAS  Google Scholar 

  35. Boyd, D. B. Electron redistribution in disulfide bonds under torsion. Theor. Chim. Acta 30, 137–150 (1973).

    Article  CAS  Google Scholar 

  36. Marszalek, P. E., Li, H., Oberhauser, A. F. & Fernandez, J. M. Chair–boat transitions in single polysaccharide molecules observed with force-ramp AFM. Proc. Natl Acad. Sci. USA 99, 4278–4283 (2002).

    Article  CAS  Google Scholar 

  37. Marszalek, P. E., Li, H. B. & Fernandez, J. M. Fingerprinting polysaccharides with single-molecule atomic force microscopy. Nature Biotechnol. 19, 258–262 (2001).

    Article  CAS  Google Scholar 

  38. Florence, T. M. Degradation of protein disulfide bonds in dilute alkali. Biochem. J. 189, 507–520 (1980).

    Article  CAS  Google Scholar 

  39. Lupton, E. M., Achenbach, F., Weis, J., Brauchle, C. & Frank, I. Molecular origins of adhesive failure: siloxane elastomers pulled from a silica surface. Phys. Rev. B 76, 125420 (2007).

    Article  Google Scholar 

  40. Lupton, E. M., Achenbach, F., Weis, J., Brauchle, C. & Frank, I. Origins of material failure in siloxane elastomers from first principles. ChemPhysChem 10, 119–123 (2009).

    Article  CAS  Google Scholar 

  41. Kruger, D., Fuchs, H., Rousseau, R., Marx, D. & Parrinello, M. Pulling monatomic gold wires with single molecules: an ab initio simulation. Phys. Rev. Lett. 89, 186402 (2002).

    Article  Google Scholar 

  42. 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  Google Scholar 

  43. Efron, B. The Jacknife, the Bootstrap and Other Resampling Plans (SIAM, 1982).

Download references

Acknowledgements

We thank L. Dougan, J. Alegre, P. Kosuri and other members of the Fernández laboratory for critical reading of the manuscript. S.G.-M. thanks the Generalitat de Catalunya for a postdoctoral fellowship through the NANO and Beatriu de Pinós programs, and also the Fundación Caja Madrid for financial support. This work was supported by NIH Grants (to J.M.F.).

Author information

Authors and Affiliations

Authors

Contributions

S.G.M. and J.M.F. conceived and designed the experiments; S.G.M., J.L., R.S. and T.K. performed the experiments; S.G.M., J.L., R.S. and T.K analysed the data; S.G.M. contributed materials and analysis tools; S.G.M and J.M.F. wrote the paper.

Corresponding authors

Correspondence to Sergi Garcia-Manyes or Julio M. Fernández.

Supplementary information

Supplementary information

Supplementary information (PDF 430 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Garcia-Manyes, S., Liang, J., Szoszkiewicz, R. et al. Force-activated reactivity switch in a bimolecular chemical reaction. Nature Chem 1, 236–242 (2009). https://doi.org/10.1038/nchem.207

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchem.207

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing