Single Molecule Studies of Protein Folding

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The objective of this project, jointly supported by Molecular Biophysics in the Division of Molecular and Cellular Biosciences and the Experimental Physical Chemistry Program in the Chemistry Division, is to investigate the protein-folding problem using single molecule studies and an optical trap (laser tweezers) methodology. Protein folding, the mechanism by which the amino acid sequence encodes the energy landscape of a protein, remains a major challenge for modern molecular biology. This project involves the use of a novel method to manipulate and monitor protein conformational changes at the single molecule level using the optical trap or laser tweezers. This apparatus allows a direct probe of the role of mechanical stress on protein structure and stability. Mechanically induced unfolding experiments have the additional advantage that, unlike bulk studies, the reaction coordinate is well defined: the end-to-end distance of the molecule. In contrast to the more common mechanical induced unfolding studies using the atomic force microscope, the softer spring constant of the optical trap offers the potential to observe protein refolding as well as unfolding. The proposed set-up consists of single proteins, or even small domains, connected to polystyrene beads via long dsDNA handles. The handles are tethered to the protein via unique disulfide bonds. One bead is held in place at the end of a pipette by suction, the other by the optical trap. Force is applied by pulling the beads away from each other. Initial studies on the well-characterized protein ribonuclease H demonstrate the ability to monitor the unfolding and refolding trajectory of a single molecule. The protein is observed to fold through a compact intermediate. The simplicity of the set-up allows for easy manipulation of proteins of varying sequence, and with various attachment points of the handles allowing the force to be applied along different regions of the structure. This project will train students at the interface of chemistry, physics, and biology. The work will require a combination of students and postdoctoral fellows with diverse backgrounds to work closely together. The project will also involve undergraduates with a background in engineering and physics, giving them direct exposure to the biological sciences.