Probing the ground state structure of the green fluorescent protein chromophore using Raman spectroscopy

We present Raman spectra, obtained using 752 nm excitation, on wild-type GF P and the S65T mutant of this intrinsically fluorescent protein together wi th data on a model chromophore, ethyl 4-(4-hydroxyphenyl)methylidene-2-meth yl-5-oxoimidazolacetate. In the pH range 1-14, the model compound has two m acroscopic pK(a)s of 1.8 and 8.2 attributed to ionization of the imidazolin one ring nitrogen and the phenolic hydroxyl group, respectively. Comparison of the model chromophore with the chromophore in wild-type GFP and the S65 T mutant reveals that the cationic form, with both the imidazolinone ring n itrogen and the phenolic oxygen protonated, is not present in these particu lar GFP proteins. Our results do not provide any evidence for the zwitterio nic form of the chromophore, with the phenolic group deprotonated and the i midazolinone ring nitrogen protonated, being present in the GFP proteins. I n addition, since the position of the Raman bands is a property exclusively of the ground state structure, the data enable us to investigate how prote in-chromophore interactions affect the ground state structure of the chromo phore without contributions from excited state effects. It is found that th e ground state structure of the anionic form of the chromophore, which is m ost relevant to the fluorescent properties, is strongly dependent on the ch romophore environment whereas the neutral form seems to be insensitive. A l inear correlation between the absorption properties and the ground state st ructure is demonstrated by plotting the absorption maxima versus the wavenu mber of a Raman band found in the range 1610-1655 cm(-1).