GFP gets flashier yet

A recent report in Chemistry & Biology (Subach et al 2010 Red fluorescent protein with reversibly photoswitchable absorbance for photochromic FRET.  Chem Biol. 17 745-55. PMID: 20659687) describes the discovery of the first red fluorescent protein that has switchable absorbance spectra. The switch is thought to happen because the chromophore undergoes a cis–trans isomerization in response to certain wavelengths of light, in this case blue or yellow light.  The switchable RFP described here changes its absorbance from an “on” state with an absorbance maximum of 567 nm to an “off” state with absorbance peaking around 440 nm.  In the “off” state, the emission intensity (at 585 nm) is also dramatically reduced, possibly because the chromophore is more flexible in this state.

One reason to be interested in such fluors is that they may add new power to in vivo FRET (Förster resonance energy transfer, a.k.a. fluorescent resonance energy transfer).  In a form of FRET called photochromic FRET (pcFRET), which had only previously been shown to be possible with photoswitchable dyes, you can arrange matters such that the donor fluor’s emissions overlap well with the acceptor fluor’s absorbance when the acceptor is in the “on” state, and overlap poorly with absorbance in the “off” state.  You can then measure the reduction in the emission from the donor when the acceptor is turned on, and see it go up again when the acceptor is turned off.  This gives you a new — possibly more accurate and sensitive — way to measure  proximity between the two fluors, instead of by using the output of the acceptor.  If your acceptor can switch reversibly and repeatedly, which this one can, then you can switch the acceptor on and off multiple times and get a more accurate measurement of fluorescence transfer by averaging the many readings.  Even if you don’t use them for FRET, you can follow biological dynamics, or label several subcellular regions one after another, or use them for super-resolution microscopy approaches such as those based on PALM (photoactivated localization microscopy).

Subach et al. were indeed able to visualize the interaction between the EGF receptor and the signaling protein Grb2 in live cells using this switchable FRET approach.  In the presence of EGF, the YFP attached to the EGF receptor shows a 20% increase in fluorescent signal when the switchable RFP attached to Grb2 is switched off, and a 20% decrease in signal when the RFP is switched on again; this difference was stable over at least 11 switching cycles. They were also able to see a clear signal for the interaction between NF-kappaB and I-kappaB.

Interestingly, a recent JACS paper (Bizzarri R, Serresi M, Cardarelli F, Abbruzzetti S, Campanini B, Viappiani C, Beltram F. 2010 Single amino acid replacement makes Aequorea victoria fluorescent proteins reversibly photoswitchable.  J Am Chem Soc. 132 85-95. PMID: 19958004) shows that a single amino acid replacement, E222Q, created reversibly photoswitchable variants of four different green/yellow GFP-derived fluorescent proteins. Bizarri et al. argued that the E222Q mutation should work based on what’s known about how the amino acids around the chromophore alter the photophysical properties of the protein, and it did.  The Subach et al. switchable protein is less elegant in that it was found by semi-directed screening, not by design, and has seven mutations, not one — none of which is E222Q.  Nevertheless, they both work, and so presumably we will see a larger variety of such switchable fluorescent proteins, with all possible desirable characteristics, in the future.  Is there no end to the extraordinary versatility of GFP?  Let’s hope not.

Subach FV, Zhang L, Gadella TW, Gurskaya NG, Lukyanov KA, & Verkhusha VV (2010). Red fluorescent protein with reversibly photoswitchable absorbance for photochromic FRET. Chemistry & Biology, 17 (7), 745-55 PMID: 20659687