Protein-DNA chimeras for single molecule mechanical folding studies with the optical tweezers (original) (raw)

Abstract

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.

Access this article

Log in via an institution

Subscribe and save

• Get 10 units per month
• Download Article/Chapter or eBook
• 1 Unit = 1 Article or 1 Chapter
• Cancel anytime Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Similar content being viewed by others

Abbreviations

AFM:

Atomic force microscope

RNase H:

E. coli ribonuclease HI

DTDP:

2,2′-Dithiodipyridine

DTT:

Dithiothreitol

RT:

Room temperature

GdmCl:

Guanidinium chloride

SDS-PAGE:

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis

HPLC:

High performance liquid chromatography

CD:

Circular dichroism

References

• Berkemeier F, Schlierf M, Rief M (2006) Mechanically controlled preparation of protein intermediates in single molecule experiments. Phys Status Solidi a-Appl Mater Sci 203:3492–3495
  Article ADS Google Scholar
• 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 81:2344–2356
  Google Scholar
• 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 10:731–737
  Article Google Scholar
• Bustamante C, Chemla YR, Forde NR, Izhaky D (2004) Mechanical processes in biochemistry. Annu Rev Biochem 73:705–748
  Article Google Scholar
• Bustamante C, Rivetti C, Keller DJ (1997) Scanning force microscopy under aqueous solutions. Curr Opin Struct Biol 7:709–716
  Article Google Scholar
• 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 10:738–743
  Article Google Scholar
• 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 96:3694–3699
  Article ADS Google Scholar
• Cecconi C, Shank EA, Bustamante C, Marqusee S (2005) Direct observation of the three-state folding of a single protein molecule. Science 309:2057–2060
  Article ADS Google Scholar
• Dabora JM, Marqusee S (1994) Equilibrium unfolding of Escherichia coli ribonuclease H: characterization of a partially folded state. Protein Sci 3:1401–1408
  Google Scholar
• Dietz H, Rief M (2006) Protein structure by mechanical triangulation. Proc Natl Acad Sci USA 103:1244–1247
  Article ADS Google Scholar
• Dietz H, Berkemeier F, Bertz M, Rief M (2006a) Anisotropic deformation response of single protein molecules. Proc Natl Acad Sci USA 103:12724–12728
  Article ADS Google Scholar
• 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 1:80–84
  Article Google Scholar
• Forman JR, Clarke J (2007) Mechanical unfolding of proteins: insights into biology, structure and folding. Curr Opin Struct Biol 17:58–66
  Article Google Scholar
• 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 93:2436–2446
  Article Google Scholar
• Graham GJ, Maio JJ (1992) A rapid and reliable method to create tandem arrays of short DNA sequences. Biotechniques 13:780–789
  Google Scholar
• Grassetti DR, Murray JF Jr (1967) Determination of sulfhydryl groups with 2,2′- or 4,4′-dithiodipyridine. Arch Biochem Biophys 119:41–49
  Article Google Scholar
• Ingber DE (2006) Cellular mechanotransduction: putting all the pieces together again. Faseb J 20:811–827
  Article Google Scholar
• Janmey PA, Weitz DA (2004) Dealing with mechanics: mechanisms of force transduction in cells. Trends Biochem Sci 29:364–370
  Article Google Scholar
• 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 127–118
  Google Scholar
• Lee G, Abdi K, Jiang Y, Michaely P, Bennett V, Marszalek PE (2006) Nanospring behaviour of ankyrin repeats. Nature 440:246–249
  Article ADS Google Scholar
• Llinas M, Marqusee S (1998) Subdomain interactions as a determinant in the folding and stability of T4 lysozyme. Protein Sci 7:96–104
  Article Google Scholar
• 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 92:3010–3021
  Article Google Scholar
• Matouschek A (2003) Protein unfolding—an important process in vivo? Curr Opin Struct Biol 13:98–109
  Article Google Scholar
• 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 106:291–295
  Article Google Scholar
• Rief M, Gautel M, Oesterhelt F, Fernandez JM, Gaub HE (1997) Reversible unfolding of individual titin immunoglobulin domains by AFM. Science 276:1109–1112
  Article Google Scholar
• Riener CK, Kada G, Gruber HJ (2002) Quick measurement of protein sulfhydryls with Ellman’s reagent and with 4,4′-dithiodipyridine. Anal Bioanal Chem 373:266–276
  Article Google Scholar
• Robic S, Berger JM, Marqusee S (2002) Contributions of folding cores to the thermostabilities of two ribonucleases H. Protein Sci 11:381–389
  Article Google Scholar
• Rounsevell R, Forman JR, Clarke J (2004) Atomic force microscopy: mechanical unfolding of proteins. Methods 34:100–111
  Article Google Scholar
• 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 93:4360–4373
  Article Google Scholar
• Smith SB, Cui Y, Bustamante C (1996) Overstretching B-DNA: the elastic response of individual double-stranded and single-stranded DNA molecules. Science 271:795–799
  Article ADS Google Scholar
• Smith SB, Cui Y, Bustamante C (2003) Optical-trap force transducer that operates by direct measurement of light momentum. Methods Enzymol 361:134–162
  Article Google Scholar
• Smith SB, Finzi L, Bustamante C (1992) Direct mechanical measurements of the elasticity of single DNA molecules by using magnetic beads. Science 258:1122–1126
  Article ADS Google Scholar
• 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):2179–2183
  Article Google Scholar
• Tskhovrebova L, Trinick J, Sleep JA, Simmons RM (1997) Elasticity and unfolding of single molecules of the giant muscle protein titin. Nature 387:308–312
  Article ADS Google Scholar
• Vogel V (2006) Mechanotransduction involving multimodular proteins: converting force into biochemical signals. Annu Rev Biophys Biomol Struct 35:459–488
  Article Google Scholar
• 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 104:7916–7921
  Article ADS Google Scholar
• 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 422:446–449
  Article ADS Google Scholar

Download references

Acknowledgments

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

Author information

Author notes

1. Ciro Cecconi
   Present address: Department of Physics, University of Modena and Reggio Emilia, Via Campi 213/A, 41100, Modena, Italy
2. Elizabeth A. Shank
   Present address: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA, 02130, USA

Authors and Affiliations

1. Department of Molecular and Cell Biology, Institute for Quantitative Biology, University of California - Berkeley, Berkeley, CA, 94720-3220, USA
   Ciro Cecconi, Elizabeth A. Shank, Susan Marqusee & Carlos Bustamante
2. Department of Chemistry, University of California, Santa Barbara, CA, 93106, USA
   Frederick W. Dahlquist
3. Department of Physics, University of California - Berkeley, Berkeley, CA, 94720, USA
   Carlos Bustamante
4. Howard Hughes Medical Institute, University of California - Berkeley, Berkeley, CA, 94720, USA
   Carlos Bustamante

Authors

1. Ciro Cecconi
   You can also search for this author inPubMed Google Scholar
2. Elizabeth A. Shank
   You can also search for this author inPubMed Google Scholar
3. Frederick W. Dahlquist
   You can also search for this author inPubMed Google Scholar
4. Susan Marqusee
   You can also search for this author inPubMed Google Scholar
5. Carlos Bustamante
   You can also search for this author inPubMed Google Scholar

Corresponding authors

Correspondence toSusan Marqusee or Carlos Bustamante.

Additional information

Ciro Cecconi and Elizabeth A. Shank contributed equally to this work.

Rights and permissions

About this article

Cite this article

Cecconi, C., Shank, E.A., Dahlquist, F.W. et al. Protein-DNA chimeras for single molecule mechanical folding studies with the optical tweezers.Eur Biophys J 37, 729–738 (2008). https://doi.org/10.1007/s00249-007-0247-y

Download citation

• Received: 31 August 2007
• Revised: 08 November 2007
• Accepted: 20 November 2007
• Published: 09 January 2008
• Issue Date: July 2008
• DOI: https://doi.org/10.1007/s00249-007-0247-y

Keywords