The Enhanced Green Fluorescent Protein as a Tool for the Analysis of Protein Dynamics and Localization: Local Fluorescence Study at the Single-molecule Level

Abstract

The green fluorescent protein (GFP) has emerged, in recent years, as a powerful reporter molecule for monitoring gene expression, protein localization and protein-protein interaction. Several mutant variants are now available differing in absorption, emission spectra and quantum yield. Here we present a detailed study of the fluorescence properties of the Phe-64-->Leu, Ser-65-->Thr mutant down to the single molecule level in order to assess its use in quantitative fluorescence microscopy and single-protein trafficking. This enhanced GFP (EGFP) is being used extensively as it offers higher-intensity emission after blue-light excitation with respect to wild-type GFP. By means of fluorescence spectroscopy we demonstrate the absence of the neutral form of the chromophore and the lack of photobleaching recovery after ultraviolet light irradiation. Furthermore, we show that the EGFP spectral properties from isolated to densely packed molecules are highly conserved. From these experiments EGFP emerges as an ideal molecule for quantitative studies of intra and intercellular tagged-protein dynamics and fluorescence-activated cell sorting, but not for monitoring single-protein trafficking over extended periods of time.

Thermogenetic neurostimulation with single-cell resolution

“…1a, Supplementary Figs 1–5). We generated two expression vectors: one encoding EGFP (refs 29, 30) downstream to the Crotalus atrox TRPA1 (caTRPA1, ref. 24) under the control of an IRES element (caTRPA1-IRES-EGFP) and the other encoding caTRPA1 fused in frame to red FP tdTomato31 via a linker containing a P2A cleavage site (caTRPA1-P2A-tdTomato) (Supplementary Fig.…”

Cis–trans photoisomerization of the chromophore in the green fluorescent protein variant E2GFP: A molecular dynamics study

“…Whereas blinking seems to be a shared feature among the various GFP mutants, no ON/OFF switching could be detected, for example, in EGFP (F64L/-S65T) under various experimental conditions [6]. However, E 2 GFP (F64L/ S65T/T203Y), i.e., EGFP with the additional mutation T203Y, is capable of ON/OFF switching [10], with peculiar features characterized in [11].…”

“…Single GFP molecules exhibit blinking fluorescence on the timescale of hundreds of ms [4][5][6][7][8]. After some cycles of blinking all investigated mutants undergo a transition to a long-lived (>1 h) dark state (the off state).…”

Single Molecule Spectroscopy of the Green Fluorescent Protein: A Critical Assessment

“…Investigations on the widely used EGFP mutant by Cinelli and coworkers and by our group showed however no optical switching behaviour for this mutant under various experimental conditions (Fig. 3) [33].…”

“…For this purpose imaging onto multichannel detectors [28][29][30][31][32][33][34], confocal microscopy [31][32][35][36], and near field scanning optical microscopy (NSOM) [37,38] were used (Fig. 2).…”