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[Preprint]. 2024 Jul 19:rs.3.rs-4638282. [Version 1] doi: 10.21203/rs.3.rs-4638282/v1

Figure 6: FP-FRET pair characterization.

Figure 6:

(A) Emission spectra of FRET constructs in which mCLIFY and YPet are used as an acceptor. CyPet-GRSMG-mCLIFY (red) and CyPet-GRSMG-YPet (blue) were excited at 433 nm. The spectra were normalized at 475 nm to emphasize the FRET signal change. (B) Emission spectra of FRET constructs in which mCLIFY and YPet were used as donors. mCLIFY-FL-mCherry (red) and YPet-FL-mCherry (blue) were excited at 517 nm. The spectra were normalized to 530 nm peak to emphasize the FRET signal change. (C) and (E) The fluorescence decay of CyPet (cyan) was fitted by two exponentials giving an average intensity weighted lifetime of 2.36 ± 0.01 ns (mean ± SD; n = 5). The decreased lifetime of CyPet in presence of an acceptor showed an average lifetime of 1.33 ± 0.03 ns (means ± SD; n = 5) for CyPet-GRSMG-mCLIFY (red, C) and 1.07 ± 0.01 ns (means ± SD; n = 5) for CyPet-GRSMG-YPet (blue, E). (D) and (F) The fluorescence lifetime of mCLIFY (cyan, D) and YPet (cyan, F) fitted with two exponentials give an average lifetime of 3.5 ± 0.01 ns (mean ± SD; n = 5) and 3.5 ± 0.001 ns (mean ± SD; n = 5) ns, respectively. The lifetime of the donor for mCLIFY-FL-mCherry (D) decreased to 2.47 ± 0.11 ns (means ± SD; n = 5) and YPet-FL-mCherry (F) to 2.33 ± 0.11 ns (means ± SD; n = 5). The FRET efficincies for all four constructs are given in SI Table 8.