The sweet protein brazzein and its interaction with the human taste receptor

项目摘要/摘要肥胖作为一个主要的公共健康问题，增加了人们对低热量天然甜味剂的兴趣。一个主要的候选者是53-残基，热稳定，蛋白质，不含碳水化合物，只有东半球灵长类动物和人类才认为是甜味的。通过诱变，我们确定了靠近N端和c端结构域的三个主要区域以及Loop43区域对异二聚体甜味受体的结合和/或活性至关重要；其他区域的突变似乎通过间接构象变化来影响甜味，这是通过核磁共振光谱检测到的。对甜受体的互补诱变研究表明，brazzein与T1R3亚基的富半胱氨酸结构域（CRD）中的特定残基相互作用。此外，建模和受体诱变研究已经在T1R2亚基的“VFTM”（维纳斯捕蝇草配体结合细胞外模块）上发现了brazzein的主要结合表面。受体上的这种大的相互作用表面区分了brazinin与小分子甜味剂的作用模式。T1R2残基在确定受体对brazzein的差异敏感性方面的具体贡献仍有待发现。我们提出三个具体目标：1。甜蛋白-受体相互作用的体外细胞测定。2. 利用核磁共振波谱技术研究巴西豆蛋白相互作用和激活甜味受体的构象和动力学要求。这些研究将有助于确定与功能特性相关的变化。3. 利用STDD-NMR技术监测brazzein及其突变体与T1R2/T1R3甜味受体及其突变体的结合。这些结果将有助于准确定义负责brazzein- sweet受体相互作用的基本分子特征，以及由此产生的信号转导无热量甜味剂作为帮助解决糖尿病和相关疾病的方法。肥胖作为一个主要的公共健康问题，增加了人们对低热量天然甜味剂的兴趣。一个主要的候选者是53-残基，热稳定，蛋白质，不含碳水化合物，只有东半球灵长类动物和人类才认为是甜味的。对brazinin -sweet受体相互作用的分子特征的解释可能会导致设计出口感更好的无热量甜味剂，作为帮助治疗糖尿病和相关疾病的方法。

DESCRIPTION (provided by applicant): Project Summary/Abstract 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.