DNA Nanotechnology pp 255-271

Part of the Methods in Molecular Biology book series (MIMB, volume 749)

DNA Molecular Handles for Single-Molecule Protein-Folding Studies by Optical Tweezers

  • Ciro Cecconi
  • Elizabeth A. Shank
  • Susan Marqusee
  • Carlos Bustamante
Protocol

Abstract

In this chapter, we describe a method that extends the use of optical tweezers to the study of the folding mechanism of single protein molecules. This method entails the use of DNA molecules as molecular handles to manipulate individual proteins between two polystyrene beads. The DNA molecules function as spacers between the protein and the beads, and keep the interactions between the tethering surfaces to a minimum. The handles can have different lengths, be attached to any pair of exposed cysteine residues, and be used to manipulate both monomeric and polymeric proteins. By changing the position of the cysteine residues on the protein surface, it is possible to apply the force to different portions of the protein and along different molecular axes. Circular dichroism and enzymatic activity studies have revealed that for many proteins, the handles do not significantly affect the folding behavior and the structure of the tethered protein. This method makes it possible to study protein folding in the physiologically relevant low-force regime of optical tweezers and enables us to monitor processes – such as refolding events and fluctuations between different molecular conformations – that could not be detected in previous force spectroscopy experiments.

Key words

Laser tweezers DNA handles Protein–DNA chimeras Single-molecule mechanical manipulation Protein folding 

References

  1. 1.
    Junker, J. P., Ziegler, F., Rief, M. (2009) Ligand-Dependent Equilibrium Fluctuations of Single Calmodulin Molecules Science 323, 633–637.CrossRefGoogle Scholar
  2. 2.
    Oberhauser, A. F., Carrion-Vazquez, M. (2008) Mechanical biochemistry of proteins one molecule at a time Journal of Biological Chemistry 283, 6617–6621.CrossRefGoogle Scholar
  3. 3.
    Borgia, A., Williams, P. M., Clarke, J. (2008) Single-molecule studies of protein folding Annual Review of Biochemistry 77, 101–125.CrossRefGoogle Scholar
  4. 4.
    Garcia-Manyes, S., Brujic, J., Badilla, C. L., Fernandez, J. M. (2007) Force-clamp spectroscopy of single-protein monomers reveals the individual unfolding and folding pathways of I27 and ubiquitin Biophysical Journal 93, 2436–2446.CrossRefGoogle Scholar
  5. 5.
    Fowler, S. B., Best, R. B., Toca Herrera, J. L., Rutherford, T. J., Steward, A., Paci, E., Karplus, M., Clarke, J. (2002) Mechanical Unfolding of a Titin Ig Domain: Structure of Unfolding Intermediate Revealed by Combining AFM, Molecular Dynamics Simulations, NMR and Protein Engineering J Mol Biol 322, 841–849.CrossRefGoogle Scholar
  6. 6.
    Forman, J. R., Clarke, J. (2007) Mechanical unfolding of proteins: insights into biology, structure and folding Current Opinion in Structural Biology 17, 58–66.CrossRefGoogle Scholar
  7. 7.
    Bustamante, C., Chemla, Y. R., Forde, N. R., Izhaky, D. (2004) Mechanical processes in biochemistry Annu Rev Biochem 73, 705–748.CrossRefGoogle Scholar
  8. 8.
    Kellermayer, M. S., Smith, S. B., Granzier, H. L., Bustamante, C. (1997) Folding-unfolding transitions in single titin molecules ­characterized with laser tweezers Science 276, 1112–1116.CrossRefGoogle Scholar
  9. 9.
    Cecconi, C., Shank, E. A., Dahlquist, F. W., Marqusee, S., Bustamante, C. (2008) Protein-DNA chimeras for single molecule mechanical folding studies with the optical tweezers European Biophysics Journal with Biophysics Letters 37, 729–738.CrossRefGoogle Scholar
  10. 10.
    Wang, M. D., Yin, H., Landick, R., Gelles, J., Block, S. M. (1997) Stretching DNA with optical tweezers Biophysical Journal 72, 1335–1346.CrossRefGoogle Scholar
  11. 11.
    Smith, S. B., Cui, Y., Bustamante, C. (1996) Overstretching B-DNA: the elastic response of individual double-stranded and single-stranded DNA molecules Science 271, 795–799.CrossRefGoogle Scholar
  12. 12.
    Cecconi, C., Shank, E. A., Bustamante, C., Marqusee, S. (2005) Direct observation of the three-state folding of a single protein molecule Science 309, 2057–2060.CrossRefGoogle Scholar
  13. 13.
    Cecconi, C., Shank, E. A., Marqusee, S., Bustamante, C. Studying protein folding with laser tweezers. In: Broglia RA, Serrano L, Tiana G, eds. Proceedings of the International School Enrico Fermi - Course CLXV: IOS Press; 2006:145–160.Google Scholar
  14. 14.
    Shank, E. A., Cecconi, C., Dill, J. W., Marqusee, S., Bustamante, C. (2010) The folding cooperativity of a protein is controlled by its chain topology Nature 465, 637–640.Google Scholar
  15. 15.
    Riener, C. K., Kada, G., Gruber, H. J. (2002) Quick measurement of protein sulfhydryls with Ellman’s reagent and with 4,4′-­dithiodipyridine Anal Bioanal Chem 373, 266–276.CrossRefGoogle Scholar
  16. 16.
    Pedersen, A. O., Jacobsen, J. (1980) Reactivity of the thiol group in human and bovine albumin at pH 3–9, as measured by exchange with 2,2’-dithiodipyridine Eur J Biochem 106, 291–295.CrossRefGoogle Scholar
  17. 17.
    Grassetti, D. R., Murray, J. F., Jr. (1967) Determination of sulfhydryl groups with 2,2’- or 4,4’-dithiodipyridine Arch Biochem Biophys 119, 41–49.CrossRefGoogle Scholar
  18. 18.
    Dietz, H., Berkemeier, F., Bertz, M., Rief, M. (2006) Anisotropic deformation response of single protein molecules Proceedings of the National Academy of Sciences of the United States of America 103, 12724–12728.CrossRefGoogle Scholar
  19. 19.
    Dietz, H., Bertz, M., Schlierf, M., Berkemeier, F., Bornschlogl, T., Junker, J. P., Rief, M. (2006) Cysteine engineering of polyproteins for single-molecule force spectroscopy Nature Protocols 1, 80–84.CrossRefGoogle Scholar
  20. 20.
    Sambrook, J., Fritsch, E. F., Maniatis, T. Molecular Cloning: A Laboratory Manual. Second Edition ed: Cold Spring Harbor Laboratory Press; 1989.Google Scholar
  21. 21.
    Smith, S. B., Cui, Y., Bustamante, C. (2003) Optical-trap force transducer that operates by direct measurement of light momentum Methods Enzymol 361, 134–162.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Ciro Cecconi
    • 1
  • Elizabeth A. Shank
  • Susan Marqusee
  • Carlos Bustamante
  1. 1.CNR-Istituto Nanoscienze S3, Department of PhysicsUniversity of Modena e Reggio EmiliaModenaItaly

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