Fluorescence resonance energy transfer between single fluorophores attached to a coiled-coil protein in aqueous solution

Fluorescence resonance energy transfer (FRET) is a technique to detect the structural changes in biomolecules. We extended this technique to the single-molecule level in aqueous solution, by combining it with total internal reflection fluorescence microscopy. Both multiple color images and fluorescence spectra at the single-molecular level were obtained to determine FRET from Cy3 to Cy5 attached to α-tropomyosin (αTm), a coiled-coil of homodimer. The FRET properties observed between single fluorophores were consistent with the premise of FRET. On excitation at the donor, the fluorescence of the donor decreased and the fluorescence of the acceptor increased. Photobleaching of one of the fluorophore affected the fluorescence of the other as predicted by the mechanism of FRET. Photobleaching occurred in a single step, confirming that the donor and acceptor fluorophores were single. Large FRET efficiency (78%) was obtained in agreement with a coiled-coil structure and the FRET decreased when the protein was denatured into two polypeptide chains. Thus, we demonstrated that it is possible to detect the assembly–disassembly of individual protein molecules as well as conformational changes occurring within a single protein molecule.