Fig. 4: Tunnel engineering of PsiR to increase the sensitivity and dynamic range.
a Four steps for PsiR response. b The potential allulose access tunnels in PsiR calculated by CAVER 3.0. The characteristics of bottleneck diameter, curvature, and flux are shown and compared. The potential allulose access tunnels in PsiR-M2, and the corresponding characteristics are compared. c The interaction energy of allulose with R109, V134, R109A, and V134A during SMD simulations. d The tunnel bottleneck diameter between sites 109 and 202 of PsiR-WT and mutants R109A, R109S, and R109G. In panel (d), the shaded bands around each curve represent the standard deviation calculated from three independent simulations. e The fluorescence values and regulatory range (fold) of different single-site PsiR mutants for 109 and 134 sites under 0/10 mM allulose. f The fluorescence values and regulatory range (fold) of combinatorial double mutants under 0/5 mM allulose. g The two-dimensional well-tempered metadynamics–extended-system adaptive biasing force (WTM-eABF) free energy of allulose access to the binding site for WT, R109G, and M2. The center-of-mass distance between allulose and D288 (d1) was defined to indicate the allulose entrance, and the center-of-mass distance between R109G or G109 and allulose (d2) was defined to indicate the interaction between allulose and R109. 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 (using WT as the control in (e) and M1 as the control in (f); P values are indicated in the panels. Source data for (b–g) are provided as a Source Data file.
