Isotopic labeling and normal-mode analysis of a model green fluorescent protein chromophore

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 νCO arising from the imidazolinone carbonyl group, whereas the bands at 1647 (cation), 1...