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The remarkable suppression of radiationless decay by the green fluorescent protein (GFP) is investigated through ultrafast fluorescence spectroscopy of its isolated chromophore in solution. Decay data are measured by fluorescence up-conversion as a function of solvent and wavelength for both neutral and anionic forms of the chromophore. All fluorescence decays are found to be well described by two exponentially decaying components. The effect of medium viscosity is slight, suggesting that the intramolecular motion promoting radiationless decay is a volume-conserving one. A minor effect of solvent polarity and H-bonding ability on the decay times is observed. The two decay constants are independent of emission wavelength, but their relative weights are not. Time-resolved fluorescence spectroscopy shows that the Stokes shift is complete in <100 fs, and that subsequent spectral evolution is limited to a small spectral narrowing. These data are discussed in terms of a two-state two-mode model, originally proposed to describe isomerization in bacteriorhodopsin (Gonzalez-Luque et al. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 9379). It is suggested that modification to the displacement and curvature of the excited-state potential energy surface of the chromophore by the protein may be sufficient to account for the dramatic enhancement of chromophore fluorescence in GFP.

Intramolecular charge transfer (ICT) processes of the neutral fluorescence probe, nile red, I, confined in the water pool of aerosol-OT (AOT) reverse micelle in n-heptane, is studied using picosecond emission spectroscopy. Utilizing the solvatochromism of nile red, only those probe molecules inside the reverse micelle are selectively excited. It is observed that while in aqueous solutions the lifetime (τf) of nile red is 650 ps, inside the reverse micelles τf increases to 3.73 ns in reverse micelle and to 2.06 ns at the highest water content of the microemulsion (w o = 32). With increase in the water-to-surfactant ratio, w o, as the water pool swells in size, the lifetime and quantum yield of emission decrease and the rate of the ICT process of nile red increases. However, the magnitude of the change (at most 8 times) in the rate of the ICT process of nile red compared to that of ordinary water is much smaller than the several thousandfold decrease observed in the solvation dynamics of water in the water pool relative to bulk water. It is proposed that while the solvation dynamics in the water pool is governed by the dielectric relaxation time, dynamics of the ICT process is controlled by the static polarity of the medium.

Solvation dynamics of 4-(dicyanomethylene)-2-methyl-6-(p-dimethylaminostyryl) 4H-pyran (DCM) in aqueous solution of a protein, human serum albumin (HSA), is studied using picosecond time-resolved emission spectroscopy. The solvation dynamics of DCM bound to HSA is found to be biexponential with one component of 600 ( 100 ps (25%) and a very long component of 10 ( 1 ns (75%). This indicates that the motion of the water molecules in the vicinity of the protein is highly constrained.

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Solvation dynamics of Coumarin 480 (C-480) in dimyristoylphosphatidylcholine (DMPC) vesicles in water is studied using picosecond time-resolved Stokes shift. In sonicated unilamellar DMPC vesicles C-480 exhibits wavelength dependent fluorescence decays. At short wavelengths a fast decay is observed while at the long wavelengths a growth in the nanosecond time scale precedes the decay. The solvation dynamics of C-480 in DMPC vesicles is found to be bimodal with two components of 0.6 and 11 ns, which is similar to the solvation dynamics of C-480 in the large water pools of AOT/n-heptane/water (AOT ) aerosol OT) microemulsions.

Solvation dynamics of Coumarin 480 (C-480) in a tetraethyl orthosilicate (TEOS) sol-gel matrix has been studied using time-resolved emission spectroscopy. In the macroscopically solid TEOS matrix the solvation dynamics of C-480 is described by a major (85%) component of 120 ( 20 ps and a minor (15%) component of 800 ( 100 ps. These components are substantially slower than the solvation dynamics in bulk water. The rotational relaxation time of C-480 in this matrix is found to be very short (<80 ps).

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