In a recent paper (http://elifesciences.org/content/early/2015/11/28/eLife.10396), it is claimed that mCherry can quench itself at high (local) concentrations. I have tried to find information about fluorescent proteins quenching themselves, but I can not find any paper or explanation, even in the refence they provide (Principles of fluorescence Spectroscopy, a book with a long chapter on quenching).
I am interested in this question because I want to make a tandem GFP (GFP fused to GFP), which I hope is twice as bright as a single GFP. If this local concentration quenching is very efficient, could the two GFP quench each other?
This is not the case in tdTomato, which is to my knowledge a dimer, or other oligomerizing fluorescent proteins.
I am aware of homo-FRET, but this should not change the fluorescene intensity, right?
I would be very thankful for any comment or advice!
Dear Christoph,
Note that quenching has to do with the surrounding microenvironment around the chromophore. In FP's, however, the chromophores are surrounded and well-protected by a rigid beta-barrel-shaped protein. If small molecules can enter the beta barrel into vicinity with the chromophore then they may induce quenching. If the specific FP is not rigid enough, then in high (global) concentration of osmolytes or crowders, it can condense, hence bring molecular groups of the internal part of the protein closer to the chromophore. Nevertheless, most FP's are quite rigid.
You ask about a local concentration increase of the same FP's. One thing I can think of is a structural change to the FP itself. One possibility is that if you fuse (as you suggest to do) two FP's, if they change their intensity, it may be caused by a structural perturbation of the FP due to the fusion. Again, this is mostly due to the relatively highly protected environment of the chromophore. Another possibility is that maybe oligomerization or misfolding occurs in high local concentration whereby the beta-barreled structure is opened/changed.
Dear Christoph,
Note that quenching has to do with the surrounding microenvironment around the chromophore. In FP's, however, the chromophores are surrounded and well-protected by a rigid beta-barrel-shaped protein. If small molecules can enter the beta barrel into vicinity with the chromophore then they may induce quenching. If the specific FP is not rigid enough, then in high (global) concentration of osmolytes or crowders, it can condense, hence bring molecular groups of the internal part of the protein closer to the chromophore. Nevertheless, most FP's are quite rigid.
You ask about a local concentration increase of the same FP's. One thing I can think of is a structural change to the FP itself. One possibility is that if you fuse (as you suggest to do) two FP's, if they change their intensity, it may be caused by a structural perturbation of the FP due to the fusion. Again, this is mostly due to the relatively highly protected environment of the chromophore. Another possibility is that maybe oligomerization or misfolding occurs in high local concentration whereby the beta-barreled structure is opened/changed.
I fully support Eitan's view. Additional, for high concentrations, there might be reabsorption of the emitted light, which leads to a decrease in the short-wavelength range of the emission spectrum (in case you look at spectra). Depending on the FP's fluorescence quantum yield, this may result in a slight decrease of the total fluorescence. However, in all practical uses (as fas as I know), FP concentrations will be far below from creating optical dense solutions (talking of absorbance values above 1)
Dear Eitan and Friedrich,
thank you very much for your great advice. Since you both indicate that there is likely no 'self-quenching' effect, I decided to do the experiment and check if GFP-GFP is indeed twice as bright as GFP in the same molecular setup. Might take a couple of month, but as soon as I have something solid I will post it here.
Thanks again!
Hi Cristoph,
I am interested to hear if you succeeded in making a double-GFP that is ~2X as bright as single GFP? I am considering the same thing and would be great to hear your experiences.
Hi Kai, currently I am writing my thesis, therefore everything is going slow and the double GFP does not have high priority for me. I hope to have it done by the end of the year.
I just stumbled about this old thread, while reading about a similar topic.
I just found that homo-FRET can actually influence the intensity of organic fluorophores (Lakowicz et al., 2003, Anal Biochem.), however in solution the quenching of fluorescent proteins seems to be minimal (Gather and Yun, 2014, Nature Comm.). Even though, I think it could be possible, if they are directly adjacent, since the Förster radius for homo-FRET of some FPs should be 3 nm or more, if I remember correctly.
I hope this helps the next one, who stumbles across this.
Dear all,
I am very interested in your discussion and I would like to have your opinion.
I would lke to know fom which density GFP or mMaple FP will quench ? is there a threshold ?
Thanks a lot in advance,
Judith
Dear Judith Miné,
I just came across your question and thought that some of the information in our recent article (https://rdcu.be/8yyM) might be useful. In there we do calculations about homo-FRET between GFP molecules in 3D ensembles. The code is open (https://github.com/CamachoDejay/FRET-calculations) and can be modified to calculate the homo-FRET for mMable FP.
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