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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 Marqusee
  • Carlos Bustamante
Original Paper

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.

Keywords

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

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

Notes

Acknowledgments

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

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Copyright information

© EBSA 2008

Authors and Affiliations

  • Ciro Cecconi
    • 1
    • 4
  • Elizabeth A. Shank
    • 1
    • 5
  • Frederick W. Dahlquist
    • 2
  • Susan Marqusee
    • 1
  • Carlos Bustamante
    • 1
    • 3
    • 6
  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

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