Molecular spectroscopy and dynamics of intrinsically fluorescent proteins:Coral red (dsRed) and yellow (Citrine)

Gene expression of intrinsically fluorescent proteins in biological systems offers new noninvasive windows into cellular function, but optimization of these probes relies on understanding their molecular spectroscopy, dynamic s, and structure. Here, the photophysics of red fluorescent protein (dsRed) from discosoma (coral), providing desired longer emission/absorption wavel engths, and an improved yellow fluorescent protein mutant (Citrine) (S65G/V 68L/Q69 M/S72A/ T203Y) for significant comparison, are characterized by usi ng fluorescence correlation spectroscopy and time-correlated single-photon counting. dsRed fluorescence decays as a single exponential with a 3.65 +/- 0.07-ns time constant, indicating a single emitting state/species independ ent of pH 4.4-9.0, in contrast with Citrine. However, laser excitation driv es reversible fluorescence flicker at 10(3)-10(4) Hz between dark and brigh t states with a constant partition fraction f(1) = 0.42 +/- 0.06 and quantu m yield of approximate to3 x 10(-3). Unlike Citrine (pKa approximate to5.7) , pH-dependent proton binding is negligible (pH 3.9-11) in dsRed. Time-reso lved anisotropy of dsRed reveals rapid depolarization (211 +/- 6 ps) plus s low rotational motion (53 +/- 8 ns), in contrast with a single rotational t ime (16 +/- 2 ns) for Citrine. The molecular dimensions, calculated from ro tational and translational diffusion, indicate that dsRed is hydrodynamical ly 3.8 +/- 0.4 times larger than predicted for a monomer, which suggests an oligomer (possibly a tetramer) configuration even at approximate to 10(-9) M. The fast depolarization is attributed to intraoligomer energy transfer between mobile nonparallel chromophores with the initial anisotropy implyin g a 24 +/- 3 degrees depolarization angle. Large two-photon excitation cros s sections (approximate to 100 GM at 990 nm for dsRed and approximate to 50 GM at 970 nm for Citrine), advantageous for two-photon-fluorescence imagin g in cells, are measured.