Single-Molecule Surface Enhanced Resonance Raman Scattering (SM-SERRS) of Green Fluorescent Proteins. PDF

(Satoshi Habuchi, Mircea Cotlet, Roel Gronheid, F.C. De Schryver, Johan Hofkens)

Single-molecule surface enhanced resonance Raman scattering (SM-SERRS) spectroscopy opens up exciting opportunities in the field of biophysics and biomedical spectroscopy. It could provide ultrasensitive detection and characterization of biophysically relevant molecules and processes as well as vibrational spectroscopy with extremely high spatial resolution. We are studying the chemical structure of the chromophore of green fluorescent proteins (GFPs) at a single-molecule level by using SM-SERRS spectroscopy.

The SERRS image of enhanced green fluorescent protein (EGFP) showed on-off blinking (see Movie 1), confirming that the Raman signal from a single EGFP molecule was detected. The chromophore of GFP can have two different forms, a protonated form and a deprotonated form (see Fig. 1). The Raman spectrum of EGFP shows major peaks at 1560 cm^-1 and 1536 cm^-1 for the protonated and the deprotonated form, respectively. The time series of the Raman spectra from one EGFP molecule displayed the sudden frequency jump from 1524 to 1562 cm^-1 (see Fig. 2). This frequency jump can be interpreted in terms of a conversion of the chromophore from the deprotonated to the protonated form. Although the conversion of the chromophore between the deprotonated and the protonated form is commonly accepted to occur in EGFP and related protein, the presence of the protonated form has been assigned on the basis of off-periods in the fluorescence of the protein in SM experiments. This hypothesis is further corroborated here at the single molecule level via the vibrational fingerprints of the protonated/deprotonated forms.

(1)

(1) Schematic illustration of the protonation/deprotonation of EGFP adsorbed on silver particles. Insets show the chemical structure of the protonated and the deprotonated form of the chromophore of EGFP.

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(2) SERRS spectra of single EGFP molecule adsorbed on a silver colloid excited at 488 nm.  Left: The time series of the spectra from two individual EGFP molecules. The bottom panels show the projection of each spectrum. Right: The same time series of the spectra as shown Left. The spectra were selected to highlight sudden spectral changes.