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利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 肥胖作为一个主要的公共健康问题，增加了人们对低卡路里、天然甜味剂的兴趣。最好的候选者是53个残留物，热稳定，蛋白质，Brazzein，不含碳水化合物，只有旧大陆灵长类动物和人类才认为是甜味。通过突变，我们确定了N-末端和C-末端结构域以及Loop43区域附近的三个主要区域对于异二聚体甜味受体的结合和/或活性是关键的；核磁共振光谱检测到，其他区域的突变似乎通过间接构象变化来影响甜味。甜味受体的互补突变研究表明，Brazzein与T1R3亚基的富含半胱氨酸的区域(CRD)中的特定残基相互作用。此外，模拟和受体诱变研究发现，在T1R2亚基的“vftm”(金蝇捕蝇剂结合胞外模块)上有一个主要的布拉泽因结合面。受体上的这种大的相互作用表面区分了巴西玉米素和小分子甜味剂的作用模式。T1R2在决定受体对罗布麻素的差异敏感性中的具体作用仍有待发现。我们提出了三个具体目标：1.甜蛋白-受体相互作用的体外细胞分析。2.利用核磁共振波谱研究辣素与甜味感受器相互作用和激活的构象和动态要求。这些研究将有助于确定与功能特性相关的变化。3.用STDD-核磁共振法检测甜蛋白T1R2/T1R3甜味受体及其突变体与Brazzein及其突变体的结合。这些结果将有助于准确地确定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非热量甜味剂，以此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.