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

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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.

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
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  2. Elizabeth A. Shank
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  3. Frederick W. Dahlquist
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  4. Susan Marqusee
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  5. Carlos Bustamante
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Corresponding authors

Correspondence to Susan 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

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  • DOI: https://doi.org/10.1007/s00249-007-0247-y

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