We present Raman spectra, obtained using 752 nm excitation, on wild-type GFP and the S65T mutant of this intrinsically fluorescent protein together with data on a model chromophore, ethyl 4-(4-hydroxyphenyl)methylidene-2-methyl-5-oxoimidazolacetate . In the pH range 1-14, the model compound has two macroscopic pK(a)s of 1.8 and 8.2 attributed to ionization of the imidazolinone ring nitrogen and the phenolic hydroxyl group, respectively. Comparison of the model chromophore with the chromophore in wild-type GFP and the S65T mutant reveals that the cationic form, with both the imidazolinone ring nitrogen and the phenolic oxygen protonated, is not present in these particular GFP proteins. Our results do not provide any evidence for the zwitterionic form of the chromophore, with the phenolic group deprotonated and the imidazolinone ring nitrogen protonated, being present in the GFP proteins. In addition, since the position of the Raman bands is a property exclusively of the ground state structure, the data enable us to investigate how protein-chromophore interactions affect the ground state structure of the chromophore without contributions from excited state effects. It is found that the ground state structure of the anionic form of the chromophore, which is most relevant to the fluorescent properties, is strongly dependent on the chromophore environment whereas the neutral form seems to be insensitive. A linear correlation between the absorption properties and the ground state structure is demonstrated by plotting the absorption maxima versus the wavenumber of a Raman band found in the range 1610-1655 cm(-1).

Unambiguous vibrational band assignments have been made to 4‘-hydroxybenzylidene-2,3-dimethyl-imidazolinone (HBDI), a model compound of the green fluorescent protein (GFP) chromophore, with the use of isotopic labeling at positions C1, C3, N4, C5, and the bridging Cα. Vibrational spectra were collected using Raman and IR spectroscopy and ab initio normal mode calculations were performed using density functional theory (DFT) and a 6-31G** basis set. Although several reports of calculations and measurements on GFP model compounds have recently appeared, we are able to definitively assign normal modes for the first time because of the use of isotopic labeling. Specifically, in the region between 1750 and 1550 cm-1, we have identified marker bands both in Raman and IR spectra for cationic, neutral, and anionic forms of the chromophore. The Raman bands at 1744 (cation), 1697 (neutral), and 1665 cm-1 (anion) are assigned to νC  O arising from the imidazolinone carbonyl group, whereas the bands at 1647 (cation), 1642 (neutral), and 1631 cm-1 (anion) are assigned to νC  C for the exocyclic CC double bond. In addition, a band at 1567 (neutral) and 1556 cm-1 (anion) is assigned to a normal mode delocalized over the imidazolinone ring and exocyclic double bond. Importantly, a band at 1582 cm-1 in cationic HBDI also involves a contribution from N−H bending of the protonated imidazolinone N4−H and consequently is very sensitive to deuteration. Because the Raman spectra of neutral and anionic HBDI in H2O and D2O are virtually identical, the sensitivity of the 1582 cm-1 band in cationic HBDI to deuteration provides a means of identifying protonation of the imidazolinone ring in green fluorescent protein. These assignments are discussed with reference to the Raman spectra of GFPs obtained in an earlier study (Bell, A. F.; He, X.; Wachter, R. M.; Tonge, P. J. Biochemistry 2000, 39, 4423−4431) and are crucial for interpreting the vibrational spectra of GFPs.

Ultrafast dispersed pump-dump-probe spectroscopy was applied to HBDI (4′-hydroxybenzylidene-2,3-dimethylimidazolinone), a model green fluorescent protein (GFP) chromophore in solution with different protonation states. The measured three-dimensional data was analyzed using a global analysis method that enables the spectral and temporal characterization of overlapping photoinduced transient states. A unified phenomenological model is presented to describe the observed data. Two excitation pathways are identified: a 1-photon excitedstate twisting and a 2-photon ionization process. The ionization pathway results in the generation of solvated electrons and HBDI radicals. The twisting dynamics was resolved on both electronic states with slower twisting on the ground state than the excited state. This is ascribed to the multidimensional hula-twist mechanism. A weak viscosity dependence was observed when the aqueous solution data were contrasted with the signals collected in a 66% glycerol/water solution.