Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
National Magnetic Resonance Facility at Madison, University of Wisconsin-Madison, Madison, WI 53706, USA.
Molecules. 2018 Oct 3;23(10):2531. doi: 10.3390/molecules23102531.
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.
味觉信号转导是一个复杂的过程，与肥胖及其相关的代谢综合征有关。甜味通过一种异源二聚体 G 蛋白偶联受体（GPCR）以种属特异性和多组织特异性的方式进行介导。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。由于难以大量生产具有均一性的味觉受体蛋白，并且缺乏可靠的体外测定方法来精确测量导致受体蛋白激活的有活性的配体结合模式，因此人类甜味受体的生物物理研究极具挑战性。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波谱（STD-NMR）结合差示扫描量热法（DSC）、圆二色性（CD）光谱和内源荧光光谱（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 中配体结合和信号转导过程的机制。
