Single-Molecule Surface-Enhanced Resonance Raman Spectroscopy of the Enhanced Green Fluorescent Protein EGFP

Abstract

We have investigated SM-SERRS spectra and intensity trajectories of individual EGFP molecules adsorbed on silver colloids. Spectral jumps, in a timescale of seconds, were observed in the SM-SERRS spectral trajectories. These jumps were attributed to a slow, reversible conversion between the protonated and deprotonated form of the chromophore in EGFP based on comparison with an ensemble Raman spectra of EGFP at different pH. The timescale of the conversion seems to match the timescale of the (long) off-periods observed in SM-fluorescence intensity trajectories. SM-SERRS intensity trajectories show, beside the slow fluctuations of the order of seconds, several faster timescales of fluctuations. We present a possible rational for those fluctuations based on literature data. We demonstrate that SM-SERRS data can be used to elucidate some of the complex photophysical processes occurring in GFPs.