Obesity as a major public health issue has increased the interest in low-calorie, natural sweeteners. A prime candidate is the 53-residue, heat stable, protein, brazzein, which contains no carbohydrate and is perceived as sweet tasting only by old world primates and humans. Through mutagenesis we have determined that three major regions near N- and C-terminal domains and Loop43 region are critical for heterodimeric sweet receptor binding and/or activity;mutations in other regions appear to affect sweetness by indirect conformational changes, as detected by NMR spectroscopy. Complementary mutagenesis studies of the sweet receptor indicate that brazzein interacts with specific residues in the cysteine-rich domain (CRD) of the T1R3 subunit. In addition, modeling and receptor mutagenesis studies have identified a major binding surface for brazzein on the """"""""VFTM"""""""" (Venus flytrap ligand binding extracellular module) of T1R2 subunit. This large interaction surface on the receptor distinguishes the mode of action of brazzein from those of small molecule sweeteners. Specific contributions of T1R2 residues in determining the differential sensitivity of the receptor to brazzein remain to be discovered. We propose three specific aims: 1. In vitro cellular assays of sweet protein-receptor interactions. 2. To use NMR spectroscopy to investigate the conformational and dynamic requirements in brazzein for its interaction and activation of the sweet taste receptor. These studies will serve to identify changes that correlate with functional properties. 3. To monitor binding of brazzein and its mutants to T1R2/T1R3 sweet receptor and its mutants by STDD-NMR spectroscopy. These results will contribute to accurately define the essential molecular features responsible for the brazzein- sweet receptor interaction and the resulting signal transduction non-caloric sweeteners as an approach to help addressing diabetes and related disorders.

Public Health Relevance

Obesity as a major public health issue has increased the interest in low-calorie, natural sweeteners. A prime candidate is the 53-residue, heat stable, protein, brazzein, which contains no carbohydrate and is perceived as sweet tasting only by old world primates and humans. Explanation of molecular features that are essential for the brazzein-sweet receptor interaction may result in design of superior tasting non-caloric sweetener as an approach to help addressing diabetes and related disorders.

Agency
National Institute of Health (NIH)
Institute
National Institute on Deafness and Other Communication Disorders (NIDCD)
Type
Research Project (R01)
Project #
5R01DC009018-02
Application #
7642409
Study Section
Somatosensory and Chemosensory Systems Study Section (SCS)
Program Officer
Davis, Barry
Project Start
2008-07-01
Project End
2013-06-30
Budget Start
2009-07-01
Budget End
2010-06-30
Support Year
2
Fiscal Year
2009
Total Cost
$325,763
Indirect Cost
Name
University of Wisconsin Madison
Department
Biochemistry
Type
Schools of Earth Sciences/Natur
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Assadi-Porter, Fariba M; Radek, James; Rao, Hongyu et al. (2018) Multimodal Ligand Binding Studies of Human and Mouse G-Coupled Taste Receptors to Correlate Their Species-Specific Sweetness Tasting Properties. Molecules 23:
Rogowski, Michael; Gollahon, Lauren; Chellini, Grazia et al. (2017) Uptake of 3-iodothyronamine hormone analogs inhibits the growth and viability of cancer cells. FEBS Open Bio 7:587-601
Singarapu, Kiran K; Tonelli, Marco; Markley, John L et al. (2016) Structure-function relationships of brazzein variants with altered interactions with the human sweet taste receptor. Protein Sci 25:711-9
Selen, Ebru Selin; Bolandnazar, Zeinab; Tonelli, Marco et al. (2015) NMR Metabolomics Show Evidence for Mitochondrial Oxidative Stress in a Mouse Model of Polycystic Ovary Syndrome. J Proteome Res 14:3284-91
Ghanian, Zahra; Maleki, Sepideh; Reiland, Hannah et al. (2014) Optical imaging of mitochondrial redox state in rodent models with 3-iodothyronamine. Exp Biol Med (Maywood) 239:151-8
Whigham, Leah D; Butz, Daniel E; Dashti, Hesam et al. (2014) Metabolic Evidence of Diminished Lipid Oxidation in Women With Polycystic Ovary Syndrome. Curr Metabolomics 2:269-278
Haviland, J A; Reiland, H; Butz, D E et al. (2013) NMR-based metabolomics and breath studies show lipid and protein catabolism during low dose chronic T(1)AM treatment. Obesity (Silver Spring) 21:2538-44
Cornilescu, Claudia C; Cornilescu, Gabriel; Rao, Hongyu et al. (2013) Temperature-dependent conformational change affecting Tyr11 and sweetness loops of brazzein. Proteins 81:919-25
Venkitakrishnan, Rani Parvathy; Benard, Outhiriaradjou; Max, Marianna et al. (2012) Use of NMR saturation transfer difference spectroscopy to study ligand binding to membrane proteins. Methods Mol Biol 914:47-63
Haviland, Julia A; Tonelli, Marco; Haughey, Dermot T et al. (2012) Novel diagnostics of metabolic dysfunction detected in breath and plasma by selective isotope-assisted labeling. Metabolism 61:1162-70

Showing the most recent 10 out of 18 publications