The temperature dependence of internal conversion in model compounds of the chromophore of the green fluorescent protein and one of its mutants has been measured. The strong temperature dependence persists in all charge forms of the model compounds, in all solvents and in a polymer matrix. The ultrafast internal conversion mechanism is thus an intrinsic property of the chromophore skeleton, rather than one of a specific charge or hydrogen-bonded form. An isoviscosity analysis shows that the coordinate which promotes internal conversion is essentially barrierless at room temperature. At reduced temperatures (or high viscosity) there is evidence for the formation of a small barrier. This may reflect a change in the nature of the microscopic solvent dynamics close to the glass transition temperature. In all cases the viscosity dependence of the rate constant for internal conversion is very weak, being approximately proportional to viscosity raised to the power of 0.25 ( 0.06. This suggests weak coupling between the relevant coordinate and macroscopic solvent viscosity. It is suggested that a potential candidate for the coordinate which promotes internal conversion is the volume-conserving "hula twist".

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Excited state relaxation in a synthetic analogue of the green fluorescent protein chromophore is investigated. Evidence is presented for rapid ground state recovery through internal conversion, with a minor channel populating a long lived bottleneck state. The rate constant for internal conversion is observed to be weakly dependent on medium viscosity over a wide range, but appears to be thermally activated. The rate constant for internal conversion does however depend on the charge of the chromophore. Some speculations on the nature of the protein chromophore interactions which might suppress this radiationless channel are made.

The electronic spectra of the chromophore of the wild type green fluorescent protein, GFP, and of a mutant form Y66F GFP in which the chromophore lacks the hydroxyl group have been studied. The acid-base properties, solvatochromism, vibronic structure and edge excitation red shift have all been measured. The results are compared with the spectra of the chromophore in the protein environment. These data suggest that the transition energy for the GFP chromophore is influenced by a number of factors in its environment, and in particular by hydrogen bonding.