Correlation of the Sweetness of Variants of the Protein Brazzein with Patterns of Hydrogen Bonds Detected by NMR Spectroscopy

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

doi:10.1074/jbc.m302663200

Cited by 30 publications

(27 citation statements)

References 49 publications

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“…Two arginines (R33 and R43) were shown to be important for brazzein’s sweetness 34. Mutant R43A was shown to exhibit profoundly reduced sweetness (only slightly sweet in powder form) 34; 42. R43 is part of the critical Site 1 loop (Loop43) further investigated here by mutagenesis, biochemical studies, and human taste trials.…”

Section: Discussionmentioning

confidence: 90%

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

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105

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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: 90%

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

Self Cite

105

106

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“…Extensive libraries of sweeteners (e.g., Spillane et al 2006) could now be examined for their ability to bind and activate human or cross-species chimeric receptors, elucidating key determinants for sweetener-receptor interactions. The relatively straightforward structural analysis of sweet protein mutants (Assadi-Porter et al 2003 ;De Simone et al 2006 ;Esposito et al 2006 ;Hobbs et al 2008 ;Spadaccini et al 2003) suggests that they may be particularly informative for understanding how changes in sweetener structure impact sweetener efficacy.…”

Section: Structure-activity Relationships For Sweeteners: Comingmentioning

confidence: 99%

The Receptor Basis of Sweet Taste in Mammals

Vigues

Dotson

Munger

2008

Results and Problems in Cell Differentiation

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The taste of sweeteners is hedonically pleasing, suggests high caloric value in food, and contributes to increased intake. In recent years, many of the molecular mechanisms underlying the detection of sweeteners have been elucidated. Of particular note is the identification of the sweet taste receptor, the heteromeric G-protein-coupled receptor T1R2:T1R3, which responds to a vast array of chemically diverse natural and artificial sweeteners. In this chapter, we discuss some of the mechanisms underlying the detection of sweeteners by mammals, with a particular focus on the function and role of the T1R2:T1R3 receptor in these processes.

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“…Mutagenesis studies suggest that the major sites of brazzein's interactions with the sweet receptor are nearby loop 43 and the N-and C-terminal regions of brazzein (Assadi-Porter, Aceti, & Markley, 2000). Mutagenesis and NMR studies have also suggested that residues 29-33 and 39-43 in the loops are involved in the sweetness of brazzein and play an important role in the interaction between brazzein and the sweet-taste receptor (Assadi-Porter, Abildgaard, Blad, & Markley, 2003;Assadi-Porter et al, 2005;Jin et al, 2003). In addition, significant contributions to brazzein's sweetness were also made nearby loop 33 and loop 9-19.…”

Section: Introductionmentioning

confidence: 96%