Last but not least, the significant redshift of the emission wavelength in the protein environment indicates the presence of a non-negligible IEF in the negative X direction. This IEF mainly originates from the charged amino acid residues surrounding the fluorophore, and mutation at these positions may result in considerable changes in the fluorescence emission maximum. The spectral tuning mechanisms discovered in this study are helpful to elucidating the key amino acid residues and the role of point mutations, as well as to the rational design of improved fluorescent proteins.