Brazzein, a Small, Sweet Protein: Effects of Mutations on its Structure, Dynamics and Functional Properties

Assadi‐Porter, Fariba M.; Abildgaard, Frits; Blad, Heike; Cornilescu, Claudia C.; Markley, John L.

doi:10.1093/chemse/bjh128

Cited by 20 publications

(9 citation statements)

References 16 publications

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“…R43 is part of the critical Site 1 loop (Loop43) further investigated here by mutagenesis, biochemical studies, and human taste trials. Our previous investigations of R43A 43 showed that, although the structure of the mutant resembles that of wild type, it is more rigid. Other mutants that exhibited reduced internal dynamics also had reduced sweetness 41.…”

Section: Discussionmentioning

confidence: 92%

Key Amino Acid Residues Involved in Multi-Point Binding Interactions between Brazzein, a Sweet Protein, and the T1R2–T1R3 Human Sweet Receptor

Assadi‐Porter

Maillet

Radek

et al. 2010

Journal of Molecular Biology

105

106

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The sweet protein brazzein activates the human sweet receptor, a heterodimeric G-protein coupled receptor (GPCR) composed of subunits T1R2 and 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 the in vitro assay. We mutated surface residues of brazzein at three putative interaction sites: Site 1 (Loop43), Site 2 (N-and C-terminus 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 Loop 9-19 (Site 3). Results from mutagenesis and chimeras of the receptor indicated that brazzein interacts with both T1R2 and T1R3 and that the Venus fly trap 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 the brazzein response. The exception, hT1R2:R217A-hT1R3, 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 in involved in subunit-subunit interaction rather than direct brazzein binding. Results from this study support a multipoint interaction between brazzein and the sweet receptor by some mechanism other than the proposed wedge models.

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Section: Discussionmentioning

confidence: 92%

Key Amino Acid Residues Involved in Multi-Point Binding Interactions between Brazzein, a Sweet Protein, and the T1R2–T1R3 Human Sweet Receptor

Assadi‐Porter

Maillet

Radek

et al. 2010

Journal of Molecular Biology

105

106

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“…A brief review of previous studies reveals that majority of investigations have been conducted for determining of factors responsible for sweetening features of the protein [7][8][9][10][11][12][13]. Among them, more studies have been performed by means of site-directed mutagenesis to identify the key residues and regions of the protein which are important in the sweetening power of Brz [9,[14][15][16][17].…”

Section: Introductionmentioning

confidence: 99%

Structural features and activity of Brazzein and its mutants upon substitution of a surfaced exposed alanine

2016

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Brazzein (Brz) is a member of sweet-tasting protein containing four disulfide bonds. It was reported as a compact and heat-resistant protein. Here, we have used site-directed mutagenesis and replaced a surface-exposed alanine with aspartic acid (A19D mutant), lysine (A19K mutant) and glycine (A19G mutant). Activity comparisons of wild-type (WT) and mutants using taste panel test procedure showed that A19G variant has the same activity as WT protein. However, introduction of a positive charge in A19K mutant led to significant increase in Brz's sweetness, while A19D has reduced sweetness compared to WT protein. Docking studies showed that mutation at position 19 results in slight chain mobility of protein at the binding surface and changing the patterns of interactions toward more effective binding of E9K variant in the concave surface of sweet taste receptor. Far-UV CD data spectra have a characteristic shape of beta structure for all variants, however different magnitudes of spectra suggest that beta-sheet structure in WT and A19G is more stable than that of A19D and A19K. Equilibrium unfolding studies with fluorescence spectroscopy and using urea and dithiothritol (DTT) as chemical denaturants indicates that A19G mutant gains more stability against urea denaturation; while conformational stability of A19D and A19K decreases when compared with WT and A19G variants. We concluded that the positive charge at the surface of protein is important factor responsible for the interaction of protein with the human sweet receptor and Ala can be considered as a key region for investigating the mechanism of the interaction of Brz with corresponding receptor.

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“…Several natural-sweet proteins have been identified and among them brazzein is a small natural protein. Brazzein is a sweet-tasting protein originally isolated and purified from the fresh fruit of Pentadiplandra brazzeana, a climbing vine that grows in Cameroon and Zaire [1][2]. Natural brazzein which consists of a single chain [3], is the smallest of the sweet-tasting proteins described to date [4][5].…”

Section: Introductionmentioning

confidence: 99%

Production of Monoclonal Specific Antibody against Brazzein Fusion Protein

Mansouri

Modarressi

2017

BJI

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Background: The use of a natural sweet protein is important as an attractive alternative to sucrose in the human diet. Rigorous research has identified the use of the artificial sweetener that leads to various diseases, cancer and heart disease. Many functional studies are necessary for examining the structure and biological activity of natural sweeteners such as Brazzein and its receptor targeting. In this report, we produced a panel of monoclonal antibodies against the recombinant brazzein which can be used for functional studies. Methods: Hybridomas cell fusion technology was used for the production of monoclonal antibody. BALB/c mice were immunized twice with purified fusion proteins with Freund adjuvant. Next, 14 days after the last injection, blood sample was collected from the eye and serum was separated and the antibody titer was detected by enzyme-linked immunosorbent assay (ELISA). The mouse with the highest antibody titers was selected. Seven days before fusion, the selected mouse was boosted with 50 µg antigen into the peritoneum to develop a robust immune response and then

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Brazzein, a Small, Sweet Protein: Effects of Mutations on its Structure, Dynamics and Functional Properties

Cited by 20 publications

References 16 publications

Key Amino Acid Residues Involved in Multi-Point Binding Interactions between Brazzein, a Sweet Protein, and the T1R2–T1R3 Human Sweet Receptor

Key Amino Acid Residues Involved in Multi-Point Binding Interactions between Brazzein, a Sweet Protein, and the T1R2–T1R3 Human Sweet Receptor

Structural features and activity of Brazzein and its mutants upon substitution of a surfaced exposed alanine

Production of Monoclonal Specific Antibody against Brazzein Fusion Protein

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