The Role of Dark States in the Photodynamics of the Green Fluorescent Protein Examined with Two-Color Fluorescence Excitation Spectroscopy

The green fluorescent protein (GFP) and its mutants are important fluorescent markers for the microscopy of biological specimens. Their photodynamics are governed by transitions between the neutral and anionic form of the light emitting chromophore. We used two-color fluorescence excitation spectroscopy to show that this is also true for mutants such as EGFP and E222Q, which in their ground state do not show any absorption attributable to the neutral chromophore. The photodynamics of E222Q are described within the framework of a 4-level system comprising two dark states. Two-color fluorescence correlation spectroscopy (FCS) has been employed to determine the rate constants for this system. The first of these states has a population rate of 3 × 105 s-1 and a lifetime of 50 μs, indicating that this is the triplet state. The second state, which we identified as the neutral chromophore, has a population rate of 4 × 105 s-1 and a lifetime of 500 μs. Our data allude to the fact that, already at low intensities, a large fraction of the molecules are in the dark states.