The low frequency vibrational modes of green fluorescent proteins

We report the observation and analysis of the low frequency vibrational modes of green fluorescent proteins (GFPs). Our study exploits the surface enhanced Raman scattering technique, which allowed the analysis of the vibrational modes of the proteins down to 300 cm(-)1. Here we present results on two GFP mutants, namely S65T/F64L GFP (EGFP) and S65T/F64L/T203Y GFP (E(2)GFP). These particularly bright mutants display almost inverted population ratio of anionic (B) to neutral (A) forms of the chromophore. By comparing the vibrational spectrum of the proteins with that of a synthetic model chromophore in solution and with the aid of first principle calculations based on density functional theory, we identify the Raman active bands in this region of frequencies. A dominant collective mode at 720 cm(-1) is found and assigned to a collective planar deformation of the chromophore. Low frequency vibrational modes belonging specifically to A and/or B structural configurations are also identified. This work demonstrates the possibility of monitoring the structural sub-states of GFPs through vibrational spectroscopy in a range of frequencies where collective modes peculiar of the double ring structure of the chromophore lie.