Does anyone know of a fluorescent protein with an emission max greater than 720 nm? Which is the best fluorescent protein with an emission max greater than 700 nm for making a BRET sensor? I have many luciferase mutants which could serve as donors in the 610-630 range provided I find a good acceptor FP in the far red range.
This table comes from a database of fluorescent proteins:
https://www.fpbase.org/table/
The highest emission wavelength listed is 720 nm.
Adam B Shapiro, Thanks so much, the table was very helpful. I noticed that FPs with an emission max of 720 nm though more red-shifted have very low quantum yields and decreased brightness. Does this mean that the luminescence output of the resulting BRET sensor would be significantly reduced? Does the quantum yield of the acceptor FP play a vital role in the efficiency of BRET?
Thank you in advance.
The BRET measurement is the ratio of fluorescence of the acceptor to luminescence of the donor. The luminescence of the donor is not dependent on the quantum yield of the acceptor, as I understand it. (I've worked with FRET, but not BRET. I assume the math is the same for the energy transfer efficiency.)
Since one of the measurements in BRET is of the acceptor emission, a low quantum yield would result in a relatively low fluorescence increase from the acceptor due to energy transfer.
The reduction in signal from donor luminescence due to energy transfer is dependent on the absorbance spectrum and extinction coefficient of the acceptor. A high extinction coefficient of the acceptor should result in a substantial decrease in the donor luminescence. The extinction coefficients of the long-wavelength fluorescent proteins in the table are pretty large, so that might offset their low quantum yields to some extent.
As far as I know, there is no theoretical reason why you have to measure the acceptor fluorescence to make a BRET measurement. You might be able to get a usable signal-to-noise ratio by measuring just the decrease in the donor luminescence, as long as there is sufficient interaction between the 2 proteins.
Adam B Shapiro, I have another question for you!
I notice that smURFP, miRFP670 and miRFP680 have similar excitation spectra and similar overlaps but smURFP is brighter and has a more significant extinction coefficient. Does this mean it will be a better acceptor of fluorescent protein for genetically encoded biosensors compared to miRFP680 or miRFP670? Assuming all three fluorescent proteins have significant overlap with the emission spectra of the donor luciferase.
Thanks in advance!
The larger extinction coefficient will make it a better acceptor, all else being equal. The other important parameters are the overlap between the emission spectrum of the donor and the absorbance spectrum of the acceptor, and the relative orientation of the dipoles of the donor and acceptor. You can measure the first parameter, but there is no way to measure the second, as far as I know. Also, if you are going to measure the fluorescence of the acceptor, its quantum yield is another important parameter.
Adam, I have been following this discussion with interest and have found your answers to very educational, thanks. I work with genetically encoded BRET sensors and have found that the design of the construct (linker length, expression of the fluorescent protein as an N-terminal or C-terminal fusion with the donor luciferase) are also important factors to be considered in design of genetically encoded BRET sensors. Do you think the size of the fluorescent protein is also an important factor and would you recommend using a smaller size acceptor FP such as using smURFP over miRFP680 or E2 crimson?
I would expect the size of the protein, and other aspects of the construct, to matter due to the fact that resonance energy transfer has a very strong sensitivity to the distance between the donor and acceptor. The closer together they are, the greater the energy transfer.
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