Two-color fluorescence correlation spectroscopy of one chromophore: Application to the E222Q mutant of the green fluorescent protein

Fluorescence correlation spectroscopy (FCS) is an important method for investigations of diffusion processes as well as of photophysical properties of fluorescing molecules. It has lately been applied in studies of the photodynamics of the green fluorescent protein (GFP). In this case FCS yields valuable information about the population of dark, non-fluorescing states of the molecule. For three-level systems rate constants into and out of the dark state can easily be determined with FCS. This task however becomes significantly more complex for molecules that possess several dark states. Here we present two-color FCS with simultaneous two-color excitation as a method that also yields spectroscopic information about the dark states. This makes the complete analysis of a molecular four-level system possible. The analysis of the GFP mutant E222Q is given as an example of two-color FCS that is readily applicable to other molecules with photoconvertible dark states. For E222Q we determine all the rate constants within the four-level system. With these data we calculate the population of the different molecular states in bulk experiments as encountered, e.g., in microscopic studies.