Fluorescence correlation spectroscopy reveals fast optical excitation-driven intramolecular dynamics of yellow fluorescent proteins

Fast excitation-driven fluctuations in the fluorescence emission of yellow- shifted green fluorescent protein mutants T203Y and T203F, with S65G/S72A, are discovered in the 10(-6)-10(-3)-s time range, by using fluorescence cor relation spectroscopy at 10(-8) M. This intensity-dependent flickering is c onspicuous at high pH, with rate constants independent of pH and viscosity with a minor temperature effect. The mean flicker rate increases linearly w ith excitation intensity for at least three decades, but the mean dark frac tion of the molecules undergoing these dynamics is independent of illuminat ion intensity over approximate to 6 x 10(2) to 5 x 10(6) W/cm(2). These res ults suggest that optical excitation establishes an equilibration between t wo molecular states of different spectroscopic properties that are coupled only via the excited state as a gateway. This reversible excitation-driven transition has a quantum efficiency of approximate to 10(-3). Dynamics of e xternal protonation, reversibly quenching the fluorescence, are also observ ed at low pH in the 10- to 100-mu s time range. The independence of these t wo bright-dark flicker processes implies the existence of at least two sepa rate dark states of these green fluorescent protein mutants. Time-resolved fluorescence measurements reveal a single exponential decay of the excited state population with 3.8-ns lifetime, after 500-nm excitation, that is pH independent. Our fluorescence correlation spectroscopy results are discusse d in terms of recent theoretical studies that invoke isomerization of the c hromophore as a nonradiative channel of the excited state relaxation.