| Abstract
| - We present Raman spectra, obtained using 752 nm excitation, on wild-type GFP and the S65Tmutant 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 compoundhas two macroscopic pKas of 1.8 and 8.2 attributed to ionization of the imidazolinone ring nitrogen andthe phenolic hydroxyl group, respectively. Comparison of the model chromophore with the chromophorein wild-type GFP and the S65T mutant reveals that the cationic form, with both the imidazolinone ringnitrogen and the phenolic oxygen protonated, is not present in these particular GFP proteins. Our resultsdo not provide any evidence for the zwitterionic form of the chromophore, with the phenolic groupdeprotonated 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 dataenable us to investigate how protein−chromophore interactions affect the ground state structure of thechromophore without contributions from excited state effects. It is found that the ground state structureof the anionic form of the chromophore, which is most relevant to the fluorescent properties, is stronglydependent on the chromophore environment whereas the neutral form seems to be insensitive. A linearcorrelation between the absorption properties and the ground state structure is demonstrated by plottingthe absorption maxima versus the wavenumber of a Raman band found in the range 1610−1655 cm-1.
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