Figure 2

FRET efficiency and maturity of ShadowG.

(a) A schematic drawing of the tandem fluorescent protein that we used to evaluate the FRET efficiency and chromophore maturation efficiency of ShadowG. XFP denotes mCherry, ShadowG, or sREACh. Amino acid sequence of the linker is shown. (b) Representative maturation efficiency images of the mEGFP-mCherry, mEGFP-ShadowG and mEGFP-sREACh tandem mutants in HeLa cells. The scale bar is 100 μm. (c) Lifetime curves of mEGFP and mEGFP-XFPs. (d) Comparison of the FRET efficiency of the mEGFP-XFP tandems. We analyzed the fluorescence lifetime decay curve averaged over the whole image (See Materials and Methods). The number of images is 20 for all conditions. Each image contains 4–13 cells and the data are presented as mean ± SEM. Asterisks denote statistical significance (p < 0.05, analysis of variance [ANOVA] followed by Scheffé’s post hoc test). (e) Maturation efficiency of individual cells was plotted in the descending order. The data are also presented as mean ± SD on the right. Asterisks denote statistical significance (p < 0.05, analysis of variance [ANOVA] followed by Scheffé’s post hoc test). (f) Inter-molecular FRET between tandem fluorescent proteins. To exclude the intra-molecular FRET, colorless mutation (Y66L) were introduced. Donor and acceptor (DNA molar ratio 1:1) were expressed in HeLa cells. The data are presented as mean ± SEM. Asterisks denote statistical significance (p < 0.05, t test).