Fig. 5: Engineering the binding pocket and allosteric sites to increase the sensitivity and dynamic range.

a The amino acids of P77, N81, F84, and M87 in the EBD. b Box plots showing the distributions of Rosetta binding energy scores (REU) for wild-type PsiR and designed variants. For each variant, n = 200 independently generated design models were evaluated in silico. Center line, median; box limits, 25–75th percentiles; whiskers, most extreme data points within 1.5×interquartile range; points denote outliers. c The fluorescence values and regulatory range (fold) of single-site mutants for P77, N81, F84, and M87 under 0/10 mM allulose. d The junction region connecting the N-terminal and C-terminal subdomains and the rigid helix region (residues 204-208) in core domain. The residues of N167 and R206 were located in this area. e The dynamic cross-correlation matrices of apo PsiR. f The dynamics cross-correlation matrices data of PsiR-complex. g The fluorescence relative to PsiR-WT and regulatory range (fold) under 0/10 mM for the saturation mutagenesis mutants of N167 and R206. h The fluorescence values and regulatory range of different single-site and combinatorial mutants under 0/2 mM allulose. i The detailed Hill equation data of PABs possessing different PsiR mutants from 0 mM to 100 mM allulose. Data are presented as mean values +/−SD (n = 3 independent experiments). Statistical analysis for panels (e) and (f) was performed by one-way ANOVA followed by Dunnett’s post hoc test; P values are indicated in the panels. Source data for (b, c, g, h, i) are provided as a Source Data file.