Fig 1.
Emission spectra of the FRET constructs.
Fluorescence emission spectrum (at 480 nm excitation) of the FRET constructs treated with thrombin. The schematics of the FRET constructs are displayed above the spectrum. "LVPR" represents the sequence GGGSLVPRGS. The decrease in the FRET in time as a result of proteolytic cleavage can be observed from the spectrum.
Fig 2.
Variations in the FRET response of FRET pairs on proteolytic cleavage over time.
The control is NowGFP-mRuby2 FRET pair without thrombin cleavage site (LVPS instead of LVPR). ΔR/R was computed as (R0-RF)/R0 where R is donor:acceptor ratio and R0 is the donor:acceptor ratio when there is no FRET and RF is the FRET ratio
Table 1.
Spectral properties of the fluorescent proteins along with the Förster radius of the FRET pairs.
Fig 3.
Fluorescence decay and fits of the FRET pairs at monitoring wavelength of 515 ns. At this monitoring wavelength only the donor decay can be observed. NowGFP Fit denotes the lifetime decay of the donor alone and FRET pair-thrombin Fit denotes the lifetime of the donor after proteolytic cleavage. The quenching of the fluorescence lifetime as a result of FRET can be observed from the decay of the FRET pairs. The lines indicate the fit and the symbols to the corresponding color of the line indicate the decay curve. The full time window of the decay and fits along with the residuals of the fits is shown in S2 Fig.
Table 2.
Fluorescence lifetime and FRET efficiency of the FRET pairs in vitro.
Fig 4.
SDS PAGE displaying the proteolytic activity.
The absence of fusion protein band and the presence of the cleaved protein band is visible from Lane 2 and 4 confirming proteolytic cleavage. The dotted arrows indicate the cleaved product and the straight arrow indicate the fusion protein band.
Fig 5.
Photobleaching of NowGFP and EGFP in E. coli cells.
Bacterial cells expressing the fluorescent proteins were excited at 488 nm laser, and the photobleaching was analyzed from images acquired using confocal microscope equipped with 63x oil objective. The difference in photobleaching of NowGFP and EGFP at different laser intensities (141 μW and 23 μW) can be observed from the figure.
Fig 6.
Intracellular FLIM of E. coli cells.
(A) Fluorescence lifetime image of the cells displaying FRET. The cells are excited at 483 nm and the selective emission from the donor was monitored through band pass filter (510/20 nm). NowGFP is the cells expressing donor alone and the variation in lifetime as a result of FRET can be observed from the cells expressing the FRET pairs. The average lifetime is calculated from approximately 30 cells. Image size is 10 μm × 10 μm. (B) Fluorescence decay curve from the cells showing FRET. The decrease in the fluorescence lifetime due to FRET can be observed from the decay curve.
Table 3.
Fluorescence lifetime and FRET efficiency of the FRET pairs inside bacterial cells (From an average of approximately 35 individual cells, for each fluorescent protein/pair).
E—FRET efficiency (calculated according to Eq 3). χ2—calculated standard weighted least squares to assess the goodness of the fit.