Multimodal Ligand Binding Studies of Human and Mouse G-Coupled Taste Receptors to Correlate Their Species-Specific Sweetness Tasting Properties
Multimodal Ligand Binding Studies of Human and Mouse G-Coupled Taste Receptors to Correlate Their Species-Specific Sweetness Tasting Properties
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DOI：
10.3390/molecules23102531
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发表时间：
2018-10-01
期刊：
影响因子：
4.6
通讯作者：
Tonelli, Marco
中科院分区：
文献类型：
--
作者：
Assadi-Porter, Fariba M.;Radek, James;Tonelli, Marco
味觉信号是一个复杂的过程，与肥胖及其相关的代谢综合征有关。甜味是通过异二聚体 G 蛋白偶联受体 (GPCR) 以物种特异性方式和多组织特异性水平介导的。甜味受体识别大量具有结构和功能多样性的配体，以调节下游信号通路的不同幅度。由于难以产生大量均一的味觉受体蛋白，并且缺乏可靠的体外测定来精确测量导致受体蛋白激活的有效配体结合模式，因此通过生物物理方法研究人类甜味受体极其困难。我们在此报告了一种多模式高通量测定法，用于监测配体结合、受体稳定性和构象变化，以模拟分子配体-受体相互作用。我们应用饱和转移差核磁共振光谱 (STD-NMR) 辅以差示扫描量热法 (DSC)、圆二色性 (CD) 光谱和本征荧光光谱 (IF) 来表征结合相互作用。我们使用互补 NMR 和生物物理分析的方法有利于研究其他 GPCR 中配体结合和信号传导过程的机制。
Taste signaling is a complex process that is linked to obesity and its associated metabolic syndromes. The sweet taste is mediated through a heterodimeric G protein coupled receptor (GPCR) in a species-specific manner and at multi-tissue specific levels. The sweet receptor recognizes a large number of ligands with structural and functional diversities to modulate different amplitudes of downstream signaling pathway(s). The human sweet-taste receptor has been extremely difficult to study by biophysical methods due to the difficulty in producing large homogeneous quantities of the taste-receptor protein and the lack of reliable in vitro assays to precisely measure productive ligand binding modes that lead to activation of the receptor protein. We report here a multimodal high throughput assay to monitor ligand binding, receptor stability and conformational changes to model the molecular ligand-receptor interactions. We applied saturation transfer difference nuclear magnetic resonance spectroscopy (STD-NMR) complemented by differential scanning calorimetry (DSC), circular dichroism (CD) spectroscopy, and intrinsic fluorescence spectroscopy (IF) to characterize binding interactions. Our method using complementary NMR and biophysical analysis is advantageous to study the mechanism of ligand binding and signaling processes in other GPCRs.