Extracting the Single-Molecule Fluorescence Trajectories of Folding Protein in Single-Pair Fluorescence Resonance Energy Transfer Experiment

Dynamic structural changes of folding protein and biological macromolecules can be monitored using the single-pair fluorescence resonance energy transfer (sp-FRET) technique, which can be used as a nanoscale ruler. Although recent progresses in applying such a state-of-the-art nanoruler to biological systems have been made, this technique still needs to be improved. In order to understand that what kind of information can be extracted from such measurements, we simulate a sp-FRET experiment as an illustrative example, in which single FRET dye pair is attached to the ends of a folding protein chain that undergoes diffusion in water. And we propose novel analysis methods with the definite purpose of extracting dynamic information at nanometer scale and the free-energy profile for protein folding as much as possible. It is suggested that the present analysis method is particularly useful for extracting the single-molecule fluorescence trajectories and free-energy landscape of protein folding. This work establishes a useful analysis technique and guideline for the sp-FRET spectroscopy experiments to monitor the folding free-energy landscape of proteins.