Modulation of the pH-dependent activity of neoculin.

(A) Human sensory test of H11X(B) and H11Y/H14A(B) variants. Panelists tasted neoculin variant solutions at pH 7.5 (white) and pH 4.0 (grey) for 15 sec and rated their sweetness levels. Each bar represents the mean ±SD (n = 4–10). The sweetness score was analyzed with a Mann-Whitney U- test (*p<0.05). X indicates D, E, F, L, N, R,Q and Y. (B) Far-UV CD spectra of the native neoculin, H11Y(B) and H11Y/H14A(B). The far-UV CD spectra were recorded in 20 mm sodium phosphate buffer, pH 7 or 20 mM sodium citrate buffer, pH 3.0. (C) The pH-dependence of the cell response to neoculin variants. WT neoculin (green), H11A(B) (pink), or H11Y(B) (cyan) (10 µM each) was applied to cells expressing hT1R2-hT1R3 and G15Gi3 under pH 5.5–7.5. The number of responsive cells was normalized relative to the maximum response to aspartame (6.7 mM) at pH 7.4. *p<0.05 and **p<0.01, one-way ANOVA with Tukey's post hoc test. Each point represents the mean ±SE from three or four independent experiments.