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.
Highlights
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
Cell-based assays suggested that the amino-terminal domain of T1R2 is required for responses to monellin, brazzein, aspartame, and neotame[13,14], the cysteine rich domain (CRD) of human T1R3 is essential for response to brazzein and thaumatin[14,15], and the amino-terminal domain of T1R3 is required for neoculin[16]
Thaumatin elicits a sweet taste at a low concentration, approximately 100,000-fold less than that of sucrose on a molar basis
Summary
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. 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. The structure of thaumatin hosts 19 acidic residues, but it is possible to restrict the choice using, as a guide, previous knowledge on the most likely region of the protein surface that interacts with the receptor[5] and preliminary results of docking studies, based on the so-called wedge model for the interaction of sweet proteins with the sweet taste receptor[9]. The wedge model remains the only viable working model to interpret the structure-activity relationship of sweet proteins
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