Single Molecule Protein Folding

Protein folding is the process in which a polypeptide folds into its characteristic and functional three-dimensional structure through possible intermediate conformational states. The stochastic nature of this process, imply that a molecule may fold through different paths. Ensemble measurements propose the disadvantage of averaging over those different paths. Single molecule spectroscopy method, on the other hand, offers detailed understanding of folding mechanism.

FRET is transfer of excitation energy from an excited donor to an acceptor flurophore. The efficiency of the energy transfer is sensetive to the distances between the flourophores, making FRET a powerful tool for studying the conformational dynamics of biomolecules.

Since the folding time scale can be in the order of seconds, immobilizing the protein for FRET measurement is essential. Our method of immobilization is to encapsulate the protein in a surface attached liposome, giving us the advantage of maintaining the protein in physiological condition.

Schematic representation of our immobilization method

A custom-made scanning microscope for automated data collection is used for acquisition of single molecule trajectory. The Emitted light from donor and acceptor fluorophores is collected separately. FRET efficiency is calculated as function of time for each molecule giving us the folding trajectory of each individual molecule. Number of intermediate states and the rate of inter-conversion is then extracted from analyzing numerous trajectories.

(a) Schematic representation of the experimental set up. (b) Typical single molecule trajectory showing acceptor and donor emission (c) FRET efficiency calculated from donor and acceptor emission.