Structure-function relationships of brazzein variants with altered interactions with the human sweet taste receptor.

Abstract

Brazzein (Brz) is a small (54 amino acid residue) sweet tasting protein with physical and taste properties superior to other non-carbohydrate sweeteners. In an investigation of sequence-dependent functional properties of the protein, we used NMR spectroscopy to determine the three-dimensional structures and dynamic properties of two Brz variants: one with a single-site substitution (D40K), which is three-fold sweeter than wild-type Brz, and one with a two-residue insertion between residues 18 and 19 (ins18 RI19 ), which is devoid of sweetness. Although the three-dimensional folds of the two variants were very similar to wild-type Brz, they exhibited local conformational and dynamic differences. The D40K substitution abolished the strong inter-stand H-bond between the side chains of residues Gln46 and Asp40 present in wild-type Brz and increased the flexibility of the protein especially at the mutation site. This increased flexibility presumably allows this site to interact more strongly with the G-protein coupled human sweet receptor. On the other hand, the Arg-Ile insertion within Loop9-19 leads to distortion of this loop and stiffening of the adjacent site whose flexibility appears to be required for productive interaction with the sweet receptor.