Photophysics and optical switching in green fluorescent protein mutants

Abstract

We demonstrate by using low-temperature high-resolution spectroscopy that red-shifted mutants of green fluorescent protein are photo-interconverted among three conformations and are, therefore, not photostable ''one-color'' systems as previously believed. From our experiments we have further derived the energy-level schemes governing the interconversion among the three forms. These results have significant implications for the molecular and cell biological applications of the green fluorescent protein family; for example, in fluorescence resonant energy transfer experiments, a change in "color" on irradiation may not necessarily be due to energy transfer but can also arise from a photo-induced conversion between conformers of the excited species.T he Aequorea victoria green fluorescent protein (GFP) has become a favored marker in molecular and cell biology because of its strong intrinsic visible fluorescence and the feasibility of fusing it to other proteins without affecting their normal functions (1-5). For example, mutants of GFP with different absorption and fluorescence spectra (4-8) are presently used in fluorescence lifetime imaging microscopy and fluorescence resonance energy transfer experiments to study protein-protein interactions, signaling, and trafficking in cellular systems (refs. 3-5 and references therein; refs. 9-12). In all of these studies it is assumed that the color changes observed in the GFP emission are caused by dynamic processes involving the proteins to which GFP is attached and do not arise in the GFP itself. That is, GFP-mutants are generally assumed to be in one conformation (i.e., to emit light of ''one color'') and remain in that conformation under laser illumination (i.e., to be photostable), a supposition that we here prove to be incorrect.The recent determination of the crystal structure of wild-type (wt) GFP and its mutants (13-15) has facilitated a structurebased, rational design of further mutants (3,5,16) in which the amino acids exchanged are either directly involved in the cyclization of the chromophore (serine 65, tyrosine 66, and glycine 67) or are located in their vicinity (4, 6-8, 17, 18). Frequently, the purpose of these mutations is to obtain one-color GFPmutants with a single conformation, in contrast to wt-GFP, which exhibits absorption bands attributed to more than one conformation (3,4,(19)(20)(21)(22)(23).The photophysics of wt-GFP presents a complex problem, and that of its mutants has not been studied in detail. The roomtemperature spectra are broad and rather unstructured (3-7, 18, 19). To determine the energy-level schemes of GFPs it is necessary to go to low temperature, where the spectrum becomes more structured. This has the additional merit that many of the thermally induced conversions are blocked at low temperature and, therefore, discrimination among individual species is facilitated. Energy-level schemes derived from low-temperature experiments put constraints on the interpretation of roomtemperature results. A recent study by high-resolution spec...

Single molecule spectroscopic characterization of GFP‐mut2 mutant for two‐photon microscopy applications

“…In contrast to blinking, which consists of a rapid switching of the fluorescence signal, the bleaching is an irreversible process (Creemers et al, 2000). For other GFP mutants, we have proved that bleaching time and blinking frequency depend on the excitation intensity (Fig.…”

“…According to TPE excitation spectra acquired through selective band-pass filters (data not shown) and to the comparison to other GFP mutants (Gregory et al, 2001), we suggest that the 820-nm excitation component corresponds to the neutral state A with emission at 454 nm and that the 885-nm component corresponds to the anionic state B, with emission at 510 nm (Creemers et al, 1999). The 760-nm excitation band is probably associated to a third excited state (state I), whose dim emission is at 540 nm (Creemers et al, 2000). In conclusion, we can observe the neutral state of the GFP-mut2 chromophore when exciting the protein at 820 nm and collecting the fluorescence output through a 460/50 (full band width ϭ 50 nm) band pass filter.…”

Spectral Versatility of Single Reef Coral Fluorescent Proteins Detected by Spectrally‐Resolved Single Molecule Spectroscopy

“…[2,4,9] Despite the widespread use of VFPs as reporters and sensors in cellular environments the versatile photophysics of fluorescent proteins is still subject to intense research. [10][11][12][13][14][15][16][17] Understanding the photophysics of these reporters is essential for accurate interpretation of the biological and biochemical processes illuminated by the fluorescent proteins. Single-pair fluorescence resonance energy transfer (spFRET) experiments designed to measure nanometer-scale protein conformational dynamics quantitatively are particularly sensitive to photophysical variations in VFPs used as donor or acceptor fluorophores.…”

Use of Chimeric Fluorescent Proteins and Fluorescence Resonance Energy Transfer to Monitor Cellular Responses

“…Furthermore, as mentioned above, steady-state FRET measurements are not always straightforward to obtain. 29 An additional possible complication derives from the spontaneous tendency of GFPs to dimerize, a feature that very much depends on where the GFPs are expressed and to what partners are fused. 20 This may not be a problem when measuring interactions between proteins in the cytosol, but when assessing interactions in a potentially crowded, 2D space such as a biological membrane, GFP oligomerization can substantially contribute to or even create the interactions observed, resulting in artifactual FRET.…”

“…However, the correct interpretation of the results obtained is not always straightforward, especially if FRET efficiency is low. 29,30 An alternative method consists of measuring FRET via donor photobleaching. 31 This technique exploits the fact that photobleaching is proportional to the excited-state lifetime of the fluorophore.…”