European Biophysics Journal

, Volume 37, Issue 6, pp 729–738 | Cite as

Protein-DNA chimeras for single molecule mechanical folding studies with the optical tweezers

  • Ciro Cecconi
  • Elizabeth A. Shank
  • Frederick W. Dahlquist
  • Susan MarquseeEmail author
  • Carlos BustamanteEmail author
Original Paper


Here we report on a method that extends the study of the mechanical behavior of single proteins to the low force regime of optical tweezers. This experimental approach relies on the use of DNA handles to specifically attach the protein to polystyrene beads and minimize the non-specific interactions between the tethering surfaces. The handles can be attached to any exposed pair of cysteine residues. Handles of different lengths were employed to mechanically manipulate both monomeric and polymeric proteins. The low spring constant of the optical tweezers enabled us to monitor directly refolding events and fluctuations between different molecular structures in quasi-equilibrium conditions. This approach, which has already yielded important results on the refolding process of the protein RNase H (Cecconi et al. in Science 309: 2057–2060, 2005), appears robust and widely applicable to any protein engineered to contain a pair of reactive cysteine residues. It represents a new strategy to study protein folding at the single molecule level, and should be applicable to a range of problems requiring tethering of protein molecules.


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



Atomic force microscope

RNase H

E. coli ribonuclease HI






Room temperature


Guanidinium chloride


Sodium dodecyl sulphate-polyacrylamide gel electrophoresis


High performance liquid chromatography


Circular dichroism



We thank members of the Marqusee and Bustamante’s labs.


  1. Berkemeier F, Schlierf M, Rief M (2006) Mechanically controlled preparation of protein intermediates in single molecule experiments. Phys Status Solidi a-Appl Mater Sci 203:3492–3495CrossRefADSGoogle Scholar
  2. Best RB, Li B, Steward A, Daggett V, Clarke J (2001) Can non-mechanical proteins withstand force? Stretching barnase by atomic force microscopy and molecular dynamics simulation. Biophys J 81:2344–2356Google Scholar
  3. Brockwell DJ, Paci E, Zinober RC, Beddard GS, Olmsted PD, Smith DA, Perham RN, Radford SE (2003) Pulling geometry defines the mechanical resistance of a beta-sheet protein. Nat Struct Biol 10:731–737CrossRefGoogle Scholar
  4. Bustamante C, Chemla YR, Forde NR, Izhaky D (2004) Mechanical processes in biochemistry. Annu Rev Biochem 73:705–748CrossRefGoogle Scholar
  5. Bustamante C, Rivetti C, Keller DJ (1997) Scanning force microscopy under aqueous solutions. Curr Opin Struct Biol 7:709–716CrossRefGoogle Scholar
  6. Carrion-Vazquez M, Li H, Lu H, Marszalek PE, Oberhauser AF, Fernandez JM (2003) The mechanical stability of ubiquitin is linkage dependent. Nat Struct Biol 10:738–743CrossRefGoogle Scholar
  7. Carrion-Vazquez M, Oberhauser AF, Fowler SB, Marszalek PE, Broedel SE, Clarke J, Fernandez JM (1999) Mechanical and chemical unfolding of a single protein: a comparison. Proc Natl Acad Sci USA 96:3694–3699CrossRefADSGoogle Scholar
  8. Cecconi C, Shank EA, Bustamante C, Marqusee S (2005) Direct observation of the three-state folding of a single protein molecule. Science 309:2057–2060CrossRefADSGoogle Scholar
  9. Dabora JM, Marqusee S (1994) Equilibrium unfolding of Escherichia coli ribonuclease H: characterization of a partially folded state. Protein Sci 3:1401–1408Google Scholar
  10. Dietz H, Rief M (2006) Protein structure by mechanical triangulation. Proc Natl Acad Sci USA 103:1244–1247CrossRefADSGoogle Scholar
  11. Dietz H, Berkemeier F, Bertz M, Rief M (2006a) Anisotropic deformation response of single protein molecules. Proc Natl Acad Sci USA 103:12724–12728CrossRefADSGoogle Scholar
  12. Dietz H, Bertz M, Schlierf M, Berkemeier F, Bornschlogl T, Junker JP, Rief M (2006b) Cysteine engineering of polyproteins for single-molecule force spectroscopy. Nat Protoc 1:80–84CrossRefGoogle Scholar
  13. Forman JR, Clarke J (2007) Mechanical unfolding of proteins: insights into biology, structure and folding. Curr Opin Struct Biol 17:58–66CrossRefGoogle Scholar
  14. Garcia-Manyes S, Brujic J, Badilla CL, Fernandez JM (2007) Force-clamp spectroscopy of single-protein monomers reveals the individual unfolding and folding pathways of I27 and ubiquitin. Biophys J 93:2436–2446CrossRefGoogle Scholar
  15. Graham GJ, Maio JJ (1992) A rapid and reliable method to create tandem arrays of short DNA sequences. Biotechniques 13:780–789Google Scholar
  16. Grassetti DR, Murray JF Jr (1967) Determination of sulfhydryl groups with 2,2′- or 4,4′-dithiodipyridine. Arch Biochem Biophys 119:41–49CrossRefGoogle Scholar
  17. Ingber DE (2006) Cellular mechanotransduction: putting all the pieces together again. Faseb J 20:811–827CrossRefGoogle Scholar
  18. Janmey PA, Weitz DA (2004) Dealing with mechanics: mechanisms of force transduction in cells. Trends Biochem Sci 29:364–370CrossRefGoogle Scholar
  19. Kellermayer MS, Smith S, Bustamante C, Granzier HL (2000) Mechanical manipulation of single titin molecules with laser tweezers. Adv Exp Med Biol 481:111–126 discussion 127–118Google Scholar
  20. Lee G, Abdi K, Jiang Y, Michaely P, Bennett V, Marszalek PE (2006) Nanospring behaviour of ankyrin repeats. Nature 440:246–249CrossRefADSGoogle Scholar
  21. Llinas M, Marqusee S (1998) Subdomain interactions as a determinant in the folding and stability of T4 lysozyme. Protein Sci 7:96–104CrossRefGoogle Scholar
  22. Manosas M, Wen JD, Li PTX, Smith SB, Bustamante C, Tinoco I, Ritort F (2007) Force unfolding kinetics of RNA using optical tweezers. II. Modeling experiments. Biophys J 92:3010–3021CrossRefGoogle Scholar
  23. Matouschek A (2003) Protein unfolding—an important process in vivo? Curr Opin Struct Biol 13:98–109CrossRefGoogle Scholar
  24. Pedersen AO, 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 106:291–295CrossRefGoogle Scholar
  25. Rief M, Gautel M, Oesterhelt F, Fernandez JM, Gaub HE (1997) Reversible unfolding of individual titin immunoglobulin domains by AFM. Science 276:1109–1112CrossRefGoogle Scholar
  26. Riener CK, Kada G, Gruber HJ (2002) Quick measurement of protein sulfhydryls with Ellman’s reagent and with 4,4′-dithiodipyridine. Anal Bioanal Chem 373:266–276CrossRefGoogle Scholar
  27. Robic S, Berger JM, Marqusee S (2002) Contributions of folding cores to the thermostabilities of two ribonucleases H. Protein Sci 11:381–389CrossRefGoogle Scholar
  28. Rounsevell R, Forman JR, Clarke J (2004) Atomic force microscopy: mechanical unfolding of proteins. Methods 34:100–111CrossRefGoogle Scholar
  29. Seol Y, Li J, Nelson PC, Perkins TT, Betterton MD (2007) Elasticity of short DNA molecules: theory and experiment for contour lengths of 0.6–7 μm. Biophys J 93:4360–4373CrossRefGoogle Scholar
  30. Smith SB, Cui Y, Bustamante C (1996) Overstretching B-DNA: the elastic response of individual double-stranded and single-stranded DNA molecules. Science 271:795–799CrossRefADSGoogle Scholar
  31. Smith SB, Cui Y, Bustamante C (2003) Optical-trap force transducer that operates by direct measurement of light momentum. Methods Enzymol 361:134–162CrossRefGoogle Scholar
  32. Smith SB, Finzi L, Bustamante C (1992) Direct mechanical measurements of the elasticity of single DNA molecules by using magnetic beads. Science 258:1122–1126CrossRefADSGoogle Scholar
  33. Steward A, Toca-Herrera JL, Clarke J (2002) Versatile cloning system for construction of multimeric proteins for use in atomic force microscopy. Protein Sci 11(9):2179–2183CrossRefGoogle Scholar
  34. Tskhovrebova L, Trinick J, Sleep JA, Simmons RM (1997) Elasticity and unfolding of single molecules of the giant muscle protein titin. Nature 387:308–312CrossRefADSGoogle Scholar
  35. Vogel V (2006) Mechanotransduction involving multimodular proteins: converting force into biochemical signals. Annu Rev Biophys Biomol Struct 35:459–488CrossRefGoogle Scholar
  36. Walther KA, Grater F, Dougan L, Badilla CL, Berne BJ, Fernandez JM (2007) Signatures of hydrophobic collapse in extended proteins captured with force spectroscopy. Proc Natl Acad Sci USA 104:7916–7921CrossRefADSGoogle Scholar
  37. Williams PM, Fowler SB, Best RB, Toca-Herrera JL, Scott KA, Steward A, Clarke J (2003) Hidden complexity in the mechanical properties of titin. Nature 422:446–449CrossRefADSGoogle Scholar

Copyright information

© EBSA 2008

Authors and Affiliations

  • Ciro Cecconi
    • 1
    • 4
  • Elizabeth A. Shank
    • 1
    • 5
  • Frederick W. Dahlquist
    • 2
  • Susan Marqusee
    • 1
    Email author
  • Carlos Bustamante
    • 1
    • 3
    • 6
    Email author
  1. 1.Department of Molecular and Cell Biology, Institute for Quantitative BiologyUniversity of California - BerkeleyBerkeleyUSA
  2. 2.Department of ChemistryUniversity of CaliforniaSanta BarbaraUSA
  3. 3.Department of PhysicsUniversity of California - BerkeleyBerkeleyUSA
  4. 4.Department of PhysicsUniversity of Modena and Reggio EmiliaModenaItaly
  5. 5.Department of Microbiology and Molecular GeneticsHarvard Medical SchoolBostonUSA
  6. 6.Howard Hughes Medical InstituteUniversity of California - BerkeleyBerkeleyUSA

Personalised recommendations