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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.

SUBMITTER: Singarapu KK

PROVIDER: S-EPMC4815422 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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

Singarapu Kiran K KK Tonelli Marco M Markley John L JL Assadi-Porter Fariba M FM

Protein science : a publication of the Protein Society 20160109 3

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 an ...[more]

PMID: 26701738

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Project description:The sweet protein brazzein [recombinant protein with sequence identical with the native protein lacking the N-terminal pyroglutamate (the numbering system used has Asp2 as the N-terminal residue)] activates the human sweet receptor, a heterodimeric G-protein-coupled receptor composed of subunits Taste type 1 Receptor 2 (T1R2) and Taste type 1 Receptor 3 (T1R3). In order to elucidate the key amino acid(s) responsible for this interaction, we mutated residues in brazzein and each of the two subunits of the receptor. The effects of brazzein mutations were assayed by a human taste panel and by an in vitro assay involving receptor subunits expressed recombinantly in human embryonic kidney cells; the effects of the receptor mutations were assayed by in vitro assay. We mutated surface residues of brazzein at three putative interaction sites: site 1 (Loop43), site 2 (N- and C-termini and adjacent Glu36, Loop33), and site 3 (Loop9-19). Basic residues in site 1 and acidic residues in site 2 were essential for positive responses from each assay. Mutation of Y39A (site 1) greatly reduced positive responses. A bulky side chain at position 54 (site 2), rather than a side chain with hydrogen-bonding potential, was required for positive responses, as was the presence of the native disulfide bond in Loop9-19 (site 3). Results from mutagenesis and chimeras of the receptor indicated that brazzein interacts with both T1R2 and T1R3 and that the Venus flytrap module of T1R2 is important for brazzein agonism. With one exception, all mutations of receptor residues at putative interaction sites predicted by wedge models failed to yield the expected decrease in brazzein response. The exception, hT1R2 (human T1R2 subunit of the sweet receptor):R217A/hT1R3 (human T1R3 subunit of the sweet receptor), which contained a substitution in lobe 2 at the interface between the two subunits, exhibited a small selective decrease in brazzein activity. However, because the mutation was found to increase the positive cooperativity of binding by multiple ligands proposed to bind both T1R subunits (brazzein, monellin, and sucralose) but not those that bind to a single subunit (neotame and cyclamate), we suggest that this site is involved in subunit-subunit interaction rather than in direct brazzein binding. Results from this study support a multi-point interaction between brazzein and the sweet receptor by some mechanism other than the proposed wedge models.

Dataset Information

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

Publications

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

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