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The green fluorescent protein is a key technology in bioimaging. In this critical review, we consider how its various applications can be tailored from knowledge of the excited state chemistry. The photophysics of the basic chromophore in solution are described in detail, and the dominant radiationless decay mechanism is characterised. The quite different photophysics of wild type GFP are described next. The unique excited state proton transfer reaction observed can be used to model proton transfer processes in proteins. Examples where the proton transfer is blocked, or redirected to occur over a low short barrier H-bond are discussed. Finally the photophysics underlying the new generation of photochemically active fluorescent proteins are discussed (155 references).

Light-driven molecular motors convert light into mechanical energy through excited-state reactions. Unidirectional rotary molecular motors based on chiral overcrowded alkenes operate through consecutive photochemical and thermal steps. The thermal (helix inverting) step has been optimized successfully through variations in molecular structure, but much less is known about the photochemical step, which provides power to the motor. Ultimately, controlling the efficiency of molecular motors requires a detailed picture of the molecular dynamics on the excited-state potential energy surface. Here, we characterize the primary events that follow photon absorption by a unidirectional molecular motor using ultrafast fluorescence up-conversion measurements with sub 50 fs time resolution. We observe an extraordinarily fast initial relaxation out of the Franck-Condon region that suggests a barrierless reaction coordinate. This fast molecular motion is shown to be accompanied by the excitation of coherent excited-state structural motion. The implications of these observations for manipulating motor efficiency are discussed.

The photodynamics of wtGFP have been studied by ultrafast time-resolved infrared spectroscopy (TIR). In addition to the expected bleaching and transient infrared absorption of bands associated with the chromophore, we observe the dynamics of the proton relay reaction in the protein. Protonation of a protein carboxylate group occurs on the tens of picoseconds time scale following photoexcitation. Comparison with data for mutant GFPs, in which excited-state proton transfer has been disabled, supports the assignment of the carboxylate to the side chain of E222, a component of the hydrogen bonding network that links the two ends of the chromophore. The TIR data show that the rate-limiting step in the proton relay is deprotonation of the chromophore.

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.

Unidirectional molecular rotary motors that harness photoinduced cis-trans (E-Z) isomerization are promising tools for the conversion of light energy to mechanical motion in nanoscale molecular machines. Considerable progress has been made in optimizing the frequency of ground-state rotation, but less attention has been focused on excited-state processes. Here the excited-state dynamics of a molecular motor with electron donor and acceptor substituents located to modify the excited-state reaction coordinate, without altering its stereochemistry, are studied. The substituents are shown to modify the photochemical yield of the isomerization without altering the motor frequency. By combining 50 fs resolution time-resolved fluorescence with ultrafast transient absorption spectroscopy the underlying excited-state dynamics are characterized. The Franck-Condon excited state relaxes in a few hundred femtoseconds to populate a lower energy dark state by a pathway that utilizes a volume conserving structural change. This is assigned to pyramidalization at a carbon atom of the isomerizing bridging double bond. The structure and energy of the dark state thus reached are a function of the substituent, with electron-withdrawing groups yielding a lower energy longer lived dark state. The dark state is coupled to the Franck-Condon state and decays on a picosecond time scale via a coordinate that is sensitive to solvent friction, such as rotation about the bridging bond. Neither subpicosecond nor picosecond dynamics are sensitive to solvent polarity, suggesting that intramolecular charge transfer and solvation are not key driving forces for the rate of the reaction. Instead steric factors and medium friction determine the reaction pathway, with the sterically remote substitution primarily influencing the energetics. Thus, these data indicate a chemical method of optimizing the efficiency of operation of these molecular motors without modifying their overall rotational frequency.

The structural dynamics following photoexcitation of a photosensing BLUF (blue light sensing using FAD) domain protein have been investigated by ultrafast transient infrared spectroscopy. Specifically, the transcriptional antirepressor AppA from Rhodobacter sphaeroides has been studied in the light and dark adapted forms and in photoactive and inactive mutants W104F and Q63L. A transient absorption has been observed at 1666 cm(-1) which is a marker mode for the photoactive state of the protein. This instantaneously formed transient is tentatively assigned to a vibrational mode of a protein residue modified through its interaction with the excited state of the chromophore. A plausible candidate consistent with the mutant studies is the carbonyl stretch of the Q63 amide side chain. These results suggest that modification of the strength of protein chromophore H-bonded interactions is the primary step in the BLUF domain photocycle. No new species were observed to be formed during the first nanosecond. Measurement of the ultrafast ground state recovery showed that the excited state of light adapted AppA is strongly quenched compared to the dark adapted state. It is proposed that the reorganization which occurs to form the signaling state is favorable to electron-transfer quenching.

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