Resonance Raman Scattering by the Green Fluorescent Protein and an Analogue of Its Chromophore

To examine the protonation state and excitation dynamics of green fluorescent protein (GFP), the electronic, resonance Raman, and Fourier transform infrared spectra of GFP were compared to the corresponding spectra of an analogue of the GFP chromophore (4-hydroxybenzylidene-2,3-dimethyl-imidazolinone, HBDI). Spectra were measured with GFP in both H2O and D2O and with HBDI in ordinary or deuterated ethanol under neutral, acidic, or basic conditions. The vibrational transitions were assigned with the aid of ab initio calculations using density functional theory methods. The results indicate that the main absorption band of GFP at 400 nm represents the neutral chromophore and the minor band at 475 nm represents the anion. The resonance Raman spectra of both GFP and neutral HBDI are dominated by a band in the region of 1565 cm-1. We assign this band to the “CN stretch” mode, which involves stretching of the imidazolinone CN bond and the central CC bond of the chromophore. The absence of resonance Raman intens...