Figure 1.
The crystal structure of neoculin (PDB ID: 2D04).
(A) NAS and NBS are colored red and blue, respectively. His residues are shown with a green stick model. NAS has two His residues at positions 14 and 36. NBS has three His residues at positions 11, 14 and 67. (B) An SDS-PAGE analysis of bacterially produced wild-type neoculin. (C) Far-UV CD spectra of bacterailly produced and native neoculin samples. The far-UV CD spectra were recorded in 20 mm sodium phosphate buffer, pH 7 or 20 mM sodium citrate buffer, pH 3.0.
Figure 2.
Dose-response relationships of His-to-Ala variants of neoculin in the cell-based assay.
The responses of hT1R2-hT1R3 and G15Gi3 transfected cells were examined after the application of WT neoculin or its single point variants at weakly acidic pH (6.0; closed circle) and at neutral pH (7.5; open circle) and to that of the 5HA variant at neutral pH (7.5; purple circle). A neoculin variant with a His-to-Ala substitution at NBS position 11 is designated H11A(B). The number of responsive cells was normalized relative to the maximum response to aspartame (6.7 mM) at pH 7.4. Each point represents the mean ±SE from three or four independent experiments.
Figure 3.
Human sensory test of His-to-Ala neoculin variants.
Panelists tasted His-to-Ala neoculin variant solutions at pH 8.0 (blue), pH 7.5 (red) and pH 4.0 (yellow) for 15 sec and rated their sweetness. Each bar represents the mean ±SD (n = 4–10).
Figure 4.
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.
Figure 5.
Docking model of neoculin and the extracellular domains of human sweet taste receptor.
NAS, NBS, hT1R3, and hT1R2 are colored red, blue, yellow, and orange, respectively. NBS His11 is shown with a green stick model.