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

摘要

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 lange, by using fluorescence correlation spectroscopy at 10~(-8) M. This intensity-dependent flickering is conspicuous at high pH, with rate constants independent of pH and viscosity with a minor temperature effect. The mean flicker rate increases linearly with excitation intensity for at least three decades, but the mean dark fraction of the molecules undergoing these dynamics is indepen- dent of illumination intensity over ≈6 × 10~2 to 5 × 10~6 W/cm~2. These results suggest that optical excitation establishes an equil- ibration between two 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 effi- ciency of ≈10~(-3). Dynamics of external protonation, reversibly quenching the fluorescence, are also observed at low pH in the 10- to 100-μs time range. The independence of these two bright-dark flicker processes implies the existence of at least two separate 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 discussed in terms of recent theoretical studies that invoke isomerization of the chromophore as a nonra- diative channel of the excited state relaxation.