title: 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.
authors.affiliation: Center for NMR And Structural Chemistry, CSIR-Indian Institute of Chemical Technology, Uppal Road, Tarnaka, Hyderabad, 500007, Telangana, India.
authors.forename: Kiran K
authors.initials: KK
authors.lastname: Singarapu
authors.affiliation: National Magnetic Resonance Facility at Madison, Biochemistry Department, University of Wisconsin-Madison, Madison, Wisconsin, 53706.
authors.forename: Marco
authors.initials: M
authors.lastname: Tonelli
authors.affiliation: National Magnetic Resonance Facility at Madison, Biochemistry Department, University of Wisconsin-Madison, Madison, Wisconsin, 53706.
authors.forename: John L
authors.initials: JL
authors.lastname: Markley
authors.affiliation: National Magnetic Resonance Facility at Madison, Biochemistry Department, University of Wisconsin-Madison, Madison, Wisconsin, 53706.|Department of Zoology, University of Wisconsin-Madison, Madison, Wisconsin, 53706.
authors.forename: Fariba M
authors.initials: FM
authors.lastname: Assadi-Porter
mesh_terms: D006801:Humans; D019684:Magnoliopsida / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D021122:Protein Subunits / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D013549:Sweetening Agents / Q000737:chemistry / Q000378:metabolism*; D013649:Taste
chemical_list: D010940:Plant Proteins; D021122:Protein Subunits; D043562:Receptors, G-Protein-Coupled; D013549:Sweetening Agents; C090521:brazzein protein, Pentadiplandra brazzeana; C431926:taste receptors, type 1
keywords: dynamics; human sweet receptor; hydrogen bonding; low calorie sweetener; nuclear magnetic resonance spectroscopy; sweet protein