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.
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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
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Acknowledgments
We thank members of the Marqusee and Bustamante’s labs.
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Author notes
- Ciro Cecconi
Present address: Department of Physics, University of Modena and Reggio Emilia, Via Campi 213/A, 41100, Modena, Italy - Elizabeth A. Shank
Present address: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA, 02130, USA
Authors and Affiliations
- 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 - Department of Chemistry, University of California, Santa Barbara, CA, 93106, USA
Frederick W. Dahlquist - Department of Physics, University of California - Berkeley, Berkeley, CA, 94720, USA
Carlos Bustamante - Howard Hughes Medical Institute, University of California - Berkeley, Berkeley, CA, 94720, USA
Carlos Bustamante
Authors
- Ciro Cecconi
You can also search for this author inPubMed Google Scholar - Elizabeth A. Shank
You can also search for this author inPubMed Google Scholar - Frederick W. Dahlquist
You can also search for this author inPubMed Google Scholar - Susan Marqusee
You can also search for this author inPubMed Google Scholar - Carlos Bustamante
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Corresponding authors
Correspondence toSusan Marqusee or Carlos Bustamante.
Additional information
Ciro Cecconi and Elizabeth A. Shank contributed equally to this work.
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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
- 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