I have two proteins: (1) a FRET control which consists in protein X tagged with GFP only and (2) a protein of interest which consists in protein X tagged both with GFP (FRET donor) and mCherry (FRET acceptor). The FRET control looks very nice and you can easily see the GFP fluorescence under the microscope. The protein of interest, on the other hand, shows an unexpectedly strong GFP quenching that, if caused entirely by FRET, would correspond to FRET efficiencies of about 70-80%. These FRET efficiencies seem to me to be too high to be real, since due to the size of the beta barrel of the fluorescent proteins I wouldn't expect the inner fluorophores to be that close to each other. When I excite mCherry and detect mCherry fluorescence I can see a very strong mCherry emission. So, the protein of interest is being expressed well and is located where it should be within the cell, but the GFP emission is just very strongly quenched. Can this quenching be due to the GFP interacting with the protein to which it is attached? Perhaps when mCherry is present GFP gets pushed inside protein X and gets quenched somehow by a distortion to the shape of the beta barrel?
Hi, in my opinion, the significant quenching of GFP is a result of FRET dominantly. The efficiency of FRET is a distance depedent, so you may try to esimate it and check for reasonabilty. While both GFP an mCherry are attached to the same protein, this distance could be very small. Distortion of barrel coud take place but the sigificant effect on fluorescence yield of chromophore located inside is quite doubtful while it stays still immobilized (at least).
Yuriy Ivanovich Glazachev Thank you very much for your answer! I'll try and analyse the results again to see if the strong donor quenching can be attributed solely to FRET. It seems unlikely due to the high efficiencies (about 75%), but maybe, as you mentioned, the FRET is real. I'll use the acceptor photobleaching methodology to confirm that the efficiencies are real.
Hi Sergio,
How did you calculate the 70-80% quenching you mentioned in your question? I'm asking since unrealistic FRET values could also result from the method.
Best,
Peter
Hi again
Actually, I also wanted to recomend you using donor photobleaching approach. However, you should keep in mind that GFP also can be bleached. Moreover, mCherry is more photostable in compare with GFP. Try to use longer wavelegnth and check for GFP bleaching.
And, I wonder, how do you really calculate the efficiency?
Hi Peter Nagy and Yuriy Ivanovich Glazachev
Thank you for your answers. I am calculating the FRET efficiency using fluorescence lifetime imaging microscopy. So, in other words, FRET efficiency = donor lifetime in the presence of the acceptor / donor lifetime in the absence of the acceptor.
Hi Sergio,
Actually the FRET efficiency should be calculated like this:
E = 1 - donor lifetime in the presence of the acceptor / donor lifetime in the absence of the acceptor.
Did you mistype the way FRET is calculated? If you calculated your 70-80% FRET efficiency with the formula you gave, the real FRET values would be 20-30%.
All the best,
Peter
Hi Peter Nagy
Thank you very much for your answer. I've just mistyped the equation. I calculate the efficiency the way you mentioned in your answer (FRET = 1 - (D/D0)).
Best regards,
Sergio.
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