A Hypersweet Protein: Removal of The Specific Negative Charge at Asp21 Enhances Thaumatin Sweetness

Abstract

Thaumatin is an intensely sweet-tasting protein that elicits sweet taste at a concentration of 50 nM, a value 100,000 times larger than that of sucrose on a molar basis. Here we attempted to produce a protein with enhanced sweetness by removing negative charges on the interacting side of thaumatin with the taste receptor. We obtained a D21N mutant which, with a threshold value 31 nM is much sweeter than wild type thaumatin and, together with the Y65R mutant of single chain monellin, one of the two sweetest proteins known so far. The complex model between the T1R2-T1R3 sweet receptor and thaumatin, derived from tethered docking in the framework of the wedge model, confirmed that each of the positively charged residues critical for sweetness is close to a receptor residue of opposite charge to yield optimal electrostatic interaction. Furthermore, the distance between D21 and its possible counterpart D433 (located on the T1R2 protomer of the receptor) is safely large to avoid electrostatic repulsion but, at the same time, amenable to a closer approach if D21 is mutated into the corresponding asparagine. These findings clearly confirm the importance of electrostatic potentials in the interaction of thaumatin with the sweet receptor.

Figure 1. Overall view of thaumatin.

Two views of thaumatin showing the relative positions of key basic residues and acidic residues chosen for mutagenesis. All the acidic residues investigated (D21, E42, D55, D59, D60, and E89) are shown in red, whereas basic ones are either in cyan (when far below the plane of panel (A), or blue when close to the surface of interaction. Panels (A,B) are related by a 90° rotation. Molecular models were generated with PyMOL.

Figure 2. Fluorescence spectra of mutant thaumatin proteins.

Plant thaumatin (black circle), D21N (red circle), E42Q (purple dot), D55N (blue triangle), D59A (green diamond), D60A (cyan diamond), E89Q (yellow square). Plant thaumatin denatured by 6 M urea (black dot) were excited at 280 nm and emission spectra were recorded at 25 °C.

Figure 3. Far-UV CD spectra of mutant thaumatin proteins.

CD spectra were recorded in 5 mM sodium phosphate buffer, pH 7.0, as described in Experimental procedures. Plant thaumatin (black dot), D21N (red dot), E42Q (purple dot), D55N (blue dot), D59A (green dot), D60A (cyan dot), E89Q (yellow dot).

Figure 4. The wedge complex of thaumatin and D21N thaumatin with the T1R2-T1R3 receptor.

(A) Two views of the complex related by a 90° rotation. The model of the receptor is shown as a line representation (blue) of the backbone whereas the model of thaumatin is shown as a neon representation of the backbone (gold). The side chains of the key basic residues of thaumatin chosen to optimize the complex are represented as thick blue neons. The corresponding side chains of the acidic residues of the receptor are represented as magenta neons. (B) Enlargement of the interaction zone surrounded by green dots in panel (A). This view shows the side of the sweet protein in contact with the receptor. Receptor residues are labeled with the prefix r2 when belonging to the T1R2 protomer and with r3 when belonging to the T1R3 protomer respectively. (C) Corresponding enlargement of the complex of D21N thaumatin with the T1R2-T1R3 receptor. The area of contact with the receptor is larger than that of wild type thaumatin. Models were built with MOLMOL.

Figure 5. Electrostatic surface of the wedge complex of D21N thaumatin with the T1R2-T1R3 receptor.

(A) Molecular model of the whole complex. (B) Molecular model of D21N thaumatin rotated and displaced to show the inner surface. (C) Molecular model of the T1R2-T1R3 receptor alone showing the interacting surface. Models were built with MOLMOL.