In this chapter, we describe a method that extends the use of optical tweezers to the study of the folding mechanism of single protein molecules. This method entails the use of DNA molecules as molecular handles to manipulate individual proteins between two polystyrene beads. The DNA molecules function as spacers between the protein and the beads, and keep the interactions between the tethering surfaces to a minimum. The handles can have different lengths, be attached to any pair of exposed cysteine residues, and be used to manipulate both monomeric and polymeric proteins. By changing the position of the cysteine residues on the protein surface, it is possible to apply the force to different portions of the protein and along different molecular axes. Circular dichroism and enzymatic activity studies have revealed that for many proteins, the handles do not significantly affect the folding behavior and the structure of the tethered protein. This method makes it possible to study protein folding in the physiologically relevant low-force regime of optical tweezers and enables us to monitor processes - such as refolding events and fluctuations between different molecular conformations - that could not be detected in previous force spectroscopy experiments.

DNA molecular handles for single-molecule protein-folding studies by optical tweezers / Cecconi, Ciro; Shank, Elizabeth A; Marqusee, Susan; Bustamante, Carlos. - 749:(2011), pp. 255-271. [10.1007/978-1-61779-142-0_18]

DNA molecular handles for single-molecule protein-folding studies by optical tweezers

CECCONI, CIRO;
2011-01-01

Abstract

In this chapter, we describe a method that extends the use of optical tweezers to the study of the folding mechanism of single protein molecules. This method entails the use of DNA molecules as molecular handles to manipulate individual proteins between two polystyrene beads. The DNA molecules function as spacers between the protein and the beads, and keep the interactions between the tethering surfaces to a minimum. The handles can have different lengths, be attached to any pair of exposed cysteine residues, and be used to manipulate both monomeric and polymeric proteins. By changing the position of the cysteine residues on the protein surface, it is possible to apply the force to different portions of the protein and along different molecular axes. Circular dichroism and enzymatic activity studies have revealed that for many proteins, the handles do not significantly affect the folding behavior and the structure of the tethered protein. This method makes it possible to study protein folding in the physiologically relevant low-force regime of optical tweezers and enables us to monitor processes - such as refolding events and fluctuations between different molecular conformations - that could not be detected in previous force spectroscopy experiments.
Methods in molecular biology (Clifton, N.J.)
DNA molecular handles for single-molecule protein-folding studies by optical tweezers / Cecconi, Ciro; Shank, Elizabeth A; Marqusee, Susan; Bustamante, Carlos. - 749:(2011), pp. 255-271. [10.1007/978-1-61779-142-0_18]
Cecconi, Ciro; Shank, Elizabeth A; Marqusee, Susan; Bustamante, Carlos
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11380/1070004
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