Li Chieh-Chin, Hung Chien-Lun, Yeh Pei-Shan, Li Chi-En, Chiang Yun-Wei
Department of Chemistry, Frontier Research Center on Fundamental and Applied Sciences of Matters, National Tsing Hua University Hsinchu 30013 Taiwan
RSC Adv. 2019 Mar 19;9(16):9014-9021. doi: 10.1039/c9ra00896a. eCollection 2019 Mar 15.
Pulsed dipolar spectroscopy (PDS) is a powerful tool to explore conformational changes of membrane proteins (MPs). However, the MPs suffer from relatively weak dipolar signals due to their complex nature in membrane environments, which consequently reduces the interspin distance resolution obtainable by PDS. Here we report the use of nanodiscs (NDs) to improve the distance resolution. Two genetically engineered membrane scaffold protein mutants are introduced, each of which is shown to form double-labeled ND efficiently and with high homogeneity. The resultant interspin distance distribution is featured by a small distribution width, suggesting high resolution. When PDS is performed on a binary mixture of the double-labeled ND devoid of MPs and the un-labeled ND with incorporated double-labeled MPs, the overall amplitude of dipolar signals is increased, leading to a critical enhancement of the distance resolution. A theoretical foundation is provided to validate the analysis. With this approach, the determination of MP structures can be studied at high resolution in NDs.
脉冲偶极光谱法（PDS）是探索膜蛋白（MPs）构象变化的有力工具。然而，由于膜蛋白在膜环境中的复杂性质，其偶极信号相对较弱，这就降低了PDS可获得的自旋间距离分辨率。在此，我们报告了使用纳米圆盘（NDs）来提高距离分辨率。引入了两种基因工程化的膜支架蛋白突变体，结果表明，每种突变体都能高效且高度均匀地形成双标记纳米圆盘。由此产生的自旋间距离分布具有较小的分布宽度，表明分辨率较高。当对不含膜蛋白的双标记纳米圆盘和掺入双标记膜蛋白的未标记纳米圆盘的二元混合物进行PDS分析时，偶极信号的整体幅度会增加，从而显著提高距离分辨率。我们提供了一个理论基础来验证该分析。通过这种方法，可以在纳米圆盘中高分辨率地研究膜蛋白结构的测定。
