Protein-DNA chimeras for single molecule mechanical folding studies with the optical tweezers
- 1.3k Downloads
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.
KeywordsLaser tweezers DNA handles Protein-DNA chimeras Single molecule mechanical manipulation Protein folding
Atomic force microscope
- RNase H
E. coli ribonuclease HI
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis
High performance liquid chromatography
We thank members of the Marqusee and Bustamante’s labs.
- 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
- Dabora JM, Marqusee S (1994) Equilibrium unfolding of Escherichia coli ribonuclease H: characterization of a partially folded state. Protein Sci 3:1401–1408Google Scholar
- Graham GJ, Maio JJ (1992) A rapid and reliable method to create tandem arrays of short DNA sequences. Biotechniques 13:780–789Google 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 127–118Google Scholar