Differential scanning fluorimetric analysis of the amino-acid binding to taste receptor using a model receptor protein, the ligand-binding domain of fish T1r2a/T1r3

Taste receptor type 1 (T1r) is responsible for the perception of essential nutrients, such as sugars and amino acids, and evoking sweet and umami (savory) taste sensations. T1r receptors recognize many of the taste substances at their extracellular ligand-binding domains (LBDs). In order to detect a wide array of taste substances in the environment, T1r receptors often possess broad ligand specificities. However, the entire ranges of chemical spaces and their binding characteristics to any T1rLBDs have not been extensively analyzed. In this study, we exploited the differential scanning fluorimetry (DSF) to medaka T1r2a/T1r3LBD, a current sole T1rLBD heterodimer amenable for recombinant preparation, and analyzed their thermal stabilization by adding various amino acids. The assay showed that the agonist amino acids induced thermal stabilization and shifted the melting temperatures (Tm) of the protein. An agreement between the DSF results and the previous biophysical assay was observed, suggesting that DSF can detect ligand binding at the orthosteric-binding site in T1r2a/T1r3LBD. The assay further demonstrated that most of the tested l-amino acids, but no d-amino acid, induced Tm shifts of T1r2a/T1r3LBD, indicating the broad l-amino acid specificities of the proteins probably with several different manners of recognition. The Tm shifts by each amino acid also showed a fair correlation with the responses exhibited by the full-length receptor, verifying the broad amino-acid binding profiles at the orthosteric site in LBD observed by DSF.

第一类味觉受体（Taste receptor type 1, T1r）负责感知糖类、氨基酸等必需营养素，并介导甜味与鲜味（浓郁风味）的味觉感受。T1r受体通过其细胞外配体结合结构域（extracellular ligand-binding domain, LBD）识别多数味觉物质。为探测环境中多样的味觉底物，T1r受体通常具备宽泛的配体特异性。然而，目前尚未有研究全面分析任意T1r LBD所覆盖的化学空间范围及其结合特性。本研究针对当前唯一可实现重组制备的T1r LBD异二聚体——青鳉T1r2a/T1r3LBD，采用差示扫描荧光法（differential scanning fluorimetry, DSF）开展实验，并通过添加多种氨基酸分析其热稳定性变化。结果显示，激动剂类氨基酸可提升蛋白热稳定性，并改变其解链温度（melting temperature, Tm）。本研究观察到DSF检测结果与既往生物物理实验结果一致，提示DSF可有效探测T1r2a/T1r3LBD正构结合位点处的配体结合事件。进一步实验表明，绝大多数受试的L-氨基酸均可引起T1r2a/T1r3LBD的解链温度偏移，而D-氨基酸无此效应，这提示该蛋白对L-氨基酸具有宽泛的特异性，且可能通过多种不同的识别模式实现结合。各氨基酸引发的解链温度偏移与全长受体的响应表现出较好的相关性，验证了DSF在LBD正构位点观测到的宽泛氨基酸结合谱。