Robust Evaluation of Intermolecular FRET Using a Large Stokes Shift Fluorophore as a Donor

Fluorescence (or Förster) resonance energy transfer (FRET) is a straightforward and sensitive technique to evaluate molecular interactions. However, most of the popular FRET pairs suffer cross-excitation of the acceptor, which could lead to false positives. To overcome this problem, we selected a large Stokes shift (LSS) fluorophore as a FRET donor. As a successful example, we employed a new FRET pair mAmetrine (an LSS yellow fluorescence protein)/DY-547 (a cyanine derivative) to substitute CFP/fluorescein that we previously employed to study molecular interactions between cyclic nucleotide-binding domains and cyclic nucleotides. The new FRET pair is practically free of cross-excitation of the acceptor. Namely, a change in the fluorescence spectral shape implies evidence of FRET without other control experiments.