THE SWEET PROTEIN BRAZZEIN AND ITS INTERACTION WITH THE HUMAN TASTE RECEPTOR

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 中心，不一定是研究者的机构。 肥胖作为一个主要的公共卫生问题，增加了对低热量天然甜味剂的兴趣。一个主要的候选者是53个残基，热稳定的蛋白质，brazzein，它不含碳水化合物，只有旧世界的灵长类动物和人类才认为是甜的。通过诱变，我们已经确定了N-和C-末端结构域附近的三个主要区域和Loop 43区域对于异源二聚体甜味受体结合和/或活性至关重要;其他区域中的突变似乎通过间接构象变化影响甜味，如通过NMR光谱检测到的。甜味受体的互补诱变研究表明，植物甜蛋白与T1 R3亚基的富含半胱氨酸结构域（CRD）中的特定残基相互作用。此外，建模和受体诱变研究已经确定了T1 R2亚基的“VFTM”（捕蝇草配体结合胞外模块）上的植物甜蛋白的主要结合表面。受体上的这种大的相互作用表面将植物甜蛋白的作用模式与小分子甜味剂的作用模式区分开来。T1 R2残基在确定受体对植物甜蛋白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.利用核磁共振技术研究甜味蛋白与甜味受体相互作用和激活甜味受体的构象和动力学要求。这些研究将有助于识别与功能特性相关的变化。3.利用STDD-NMR技术监测植物甜蛋白及其突变体与T1 R2/T1 R3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-甜味受体相互作用的基本分子特征以及由此产生的无热量甜味剂的信号转导，作为帮助解决糖尿病和相关疾病的方法。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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 distiguishes the mode of action of brazzein from those of small molecule sweeteners. Specific contributions of T1R2 resdidues 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 fuctional 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 address diabetes and related disorders.