固定化O6-烷基鸟嘌呤-DNA甲基转移酶标签重组蛋白构象表征新方法的建立及应用

本项目针对现有固定化蛋白质构象表征方法不能给出蛋白质活性信息的问题，以G-蛋白偶联受体超家族成员β2-肾上腺素受体（β2-AR）为例，将O6-烷基鸟嘌呤-DNA甲基转移酶重组至受体的C-末端，利用酶的特异性烷基转移活性，将受体定向固定在苄基鸟嘌呤修饰的树脂表面，建立β2-AR色谱新模型；以激活型G-蛋白（Gs-蛋白）为构象探针，依据受体与Gs-蛋白结合强度与受体构象定量相关的特性，用色谱法测定不同药物配体诱导后受体与Gs-蛋白结合过程动力学、热力学及超热力学参数，用飞行时间-二次离子质谱测定不同构象态受体表面氨基酸残基种类和强度，非线性拟合法获得色谱参数与质谱参数间的相关性，建立能基于固定化蛋白质信号传导活性的构象表征方法学；用该方法筛选中药白芥子的活性成分，建立能区分激动和拮抗活性的药物成分筛选方法学。该研究能为其他固定化蛋白质构象表征提供新思路，对高效创新药物的开发具有重要的实际意义。

Several technologies have been developed for characterizing the conformation of immobilzed protein in previous reports. It is notable that these assays are not capable of providing any bioactive informations of the protein. To address this issue, this proposal is suggested using β2-adrenoceptor (β2-AR), a member of G-protein coupled receptors superfamily, as a probe. Firstly, O6-alkylguanine-DNA alkyltransferase is fused into the C-terminal of β2-AR to oriented immoblize the receptor onto the surface of resin using the sepcific alkyl-tranferring bioactivity of the enzenmy. The immobilized protein is used to establish β2-AR chromatography. Secondly, the chromatography is utilized to determine the kinetic, themodynamic and ultra-thermodynamic behaviors of the interaction between β2-AR and Gs-protein. Moreover, the fingerprint of the aminol groups on the surface of the recptor is also characterized by time of flight-seondary ion-mass spectrometry. Nonlinear regression is used to reveal the relationship between the parameters collected by chromatography and mass spectrometry to develop a new methodology for bioactivley characterize the conformation of immobilized protein. The proposed method is finally applied in screening the bioactive compounds in Semen sinapis to construct a screening method with the properties of recognizing agonists and antagonists. This proposal is able to provide strategy for characterizing conformation of other proteins. It plays particularly important role in innovative drug discovery.