FIG. 1.

Design and engineering of highly responsive FRET-based sensors with optimized linkers. (A) Primary structures of N- and C-terminal composite linkers consisting of various connecting peptides and flanking domains. The suffixes to the sensor names indicate different types of linkers (see Fig. S3A in the supplemental material). (B) Ratio changes induced by maltose are shown, where solid and dashed bars represent 530/480 emission ratios in the absence and presence, respectively, of 10 mM maltose. Values on top of the bars indicate the levels of increase or reduction in emission intensity ratios. Data are presented as the averages of at least three experiments. (C) Maltose titration curves for maltose sensors with optimized linkers. The solid curve in each case represents the least-squares fit of ratio change to concentration of maltose, as described in Materials and Methods. The inset shows the predicted helical structure of the linker₁ moiety, consisting of a connecting peptide LH and flanking regions of CFP and MBP. Color key: main body of CFP (cyan), terminal 3₁₀-helix extended from CFP (red), connecting peptide LH (yellow), and MBP (purple).