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 #
5R01DC009018-05
Application #
8277802
Study Section
Somatosensory and Chemosensory Systems Study Section (SCS)
Program Officer
Sullivan, Susan L
Project Start
2008-07-01
Project End
2014-06-30
Budget Start
2012-07-01
Budget End
2014-06-30
Support Year
5
Fiscal Year
2012
Total Cost
$312,185
Indirect Cost
$68,913
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
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