Two kinase sensors (reporters) have been developed to detect FGFR activity based
ID: 81089 • Letter: T
Question
Two kinase sensors (reporters) have been developed to detect FGFR activity based on FRET generation, and their domain structures are shown below. Both sensors have been confirmed to be highly specific for FGFR (i.e., no other kinases can activate either sensor). When the sensors are expressed in cells, stimulation of the cells with FGF leads to the two activation profiles shown below.
(a) Which sensor is more likely to report true FGFR activation profile in the cell?
(b) What elements in the sensors may have contributed to the observed difference (and how)?
Sensor 1 Sensor 2 CFP SH20a) pTyr motif 1 YFP CFP SH2(b) pTyr motif 2 YFP UL 20 min 20 min 10 min 10 min Time after FGF stimulation Time after FGF stimulationExplanation / Answer
Assuming both sensor have been confirmed to be highly specific for FGFR.
Firstly FRET stands for, Fluorescence resonance energy transfer which involves the radiation less transfer of energy from an excited donor fluorophore to an acceptor fluorophore. This technique is used for studying molecular interaction of living cells. And FGFR stands for, Fibroblast Growth Factor Receptor which is critical for many physiological roles in the body genesis.
The Donor and Acceptor fluorophores are autofluorescent proteins, here the CFP and YFP. Further the best sensory results are those where there is sufficient separation in excitation spectra for selective activation of donor fluorophore, which tends to give overlap between the emission spectrum of the donor and excitation spectrum of acceptor to obtain efficient energy transfer.
So here from above two graphs of FRET efficiency it is clear that the Sensor 1 has better separation of emission spectra between donor and acceptor fluorophores. So the answer is:
(a) Sensor 1 is more likely to report true FGFR activation profile in cell.
(b) The observed difference is mainly due to the CFP and YFP (the auto-fluorescent protein) separation in excitation spectra for selective stimulation of the donor protein and overlap between the emission spectrum of donor and excitation spectrum of acceptor for efficient energy transmission.
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