Applying three-dimensional fluorescence spectroscopy to reveal a new fluorescence peak of the Enhanced Green Fluorescent Protein

The Enhanced Green Fluorescent Protein (EGFP) has a natural and intense fluorescence, which is very promising for medical applications. The first fluorescence studies on EGFP suggested that the protein had a single form and fluorescence peak, in contrast to what was seen for its original wild-type. However, after the experimental determination of the crystalline structure of EGFP, multiple groups reported two different conformations [1-2]. To clarify the controversy regarding the EGFP homogeneous fluorescence spectrum, this study evaluated the EGFP 3D fluorescence spectra at lower wavelengths and under different conditions. The discovery of a new fluorescence peak helped to elucidate the contradiction regarding the two conformations of EGFP. It was also shown that the intensity of the second peak was pH dependent, sensitive to high temperatures and linearly related to EGFP concentration. It also highlights that 3D spectra are a powerful tool for the discovery of elusive fluorophores.