Intrinsic Fluorescence Fluctuations of Large Stokes Shift Fluorescent Proteins: LSSmOrange and mKeima Studied by Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy

Large Stokes shift fluorescent protein (LSS FP) is a promising tool for biological research that is compatible with a broad range of advanced microscopy techniques. Here, we have investigated the ground-state heterogeneity and dynamics of two LSS FPs, LSSmOrange and mKeima, by two-dimensional fluorescence lifetime correlation spectroscopy. Both LSS FPs were found to have two states with distinct mean fluorescence lifetimes. From the pH-dependent change of relative populations, the states with short and long fluorescence lifetimes were assigned to the cis - and trans - forms of the chromophore, respectively. These states showed interconversion on two distinct time scales, submicroseconds to a few microseconds and submilliseconds to milliseconds. Excitation-power dependence and the symmetry of cross-correlations indicated that while the microsecond dynamics is thermally induced, the slow submillisecond dynamics is an irreversible photoinduced process. The knowledge obtained is expected to be helpful in the spectroscopic and imaging applications of LSS FPs.