Single Molecule SERS Leads to the Observation of the Intermediate Form of Green Fluorescent Protein Chromophore

The protonated (P) form of the green fluorescent protein (GFP) involves a protolytic reaction under violet-blue light by forming an excited anion of the intermediate form of the chromophore from the photo-excited protonated form. This study reports the observation of this proton movement cycle by acquiring vibrational spectra of single GFP molecules using "nanometal-on-semiconductor" SERS substrates and applying a "pump and probe" technique. A new peak at 1510 cm(-1) prominently exists with 1560 cm(-1)(P) peak. Statistical analysis of the GFP population reveals a higher probability (about 9 times) of observing both 1510 cm(-1) and 1560 cm(-1) peak over the probability of finding both 1510 cm(-1) and 1530 cm(-1) (deprotonated) in one 'spectral jump'. We recognize this unique Raman peak at 1510 cm(-1) as a marker for the intermediate form of the chromophore. This peak is also observed both in 'Cis' (1260 cm(-1)) and 'Trans' (1280 cm(-1)) configuration of the chromophore, but considerably more with 'Cis' configuration.