水通道蛋白AQPs质子排斥机理是AQPs的核心科学问题。Fujiyoshi基于磷脂膜中解析的3.8Å AQP1结构提出与NPA基序两个保守Asn同时结合的中心水分子在质子排斥中发挥了关键作用。但该结构的分辨率不足以观察到水分子与Asn的结合方式。Neutze在去垢剂中解析的0.88Å Aqy1结构中观察到两个Asn分别与两个独立的水分子结合。然而，环境会影响水分子在AQPs水道中的分布，需要在更接近AQPs生理环境的磷脂膜中检查Asn与水分子的结合方式。17O固体NMR是磷脂膜中研究水分子和膜蛋白相互作用的有力工具。本项目拟用15N选择性标记Asn、17O标记水，通过DNP增强的固体NMR实验D-HMQC、ZF-TEDOR和3QMAS分别观测15N-17O相关性、核间距和17O四极耦合常数，结合量子化学计算，揭示AqpZ中Asn与水分子的结合方式，阐明磷脂膜中AQPs质子排斥机理。

The proton repulsion mechanism of aquaporins (AQPs) is the core scientific question of AQPs. Based on the 3.8Å AQP1 structure resolved in the phospholipid membrane, Fujiyoshi proposed that the central water molecule that simultaneously binds to two conservative Asn residues of the NPA motif plays a key role in proton repulsion. Unfortunately, it is difficult to directly observe the binding mode of water molecules and Asn residues because of the poor resolution of this structure. Neutze clearly observed two Asn residues respectively bound to two independent water molecules in the 0.88Å Aqy1 structure resolved in the detergent. However, the environment affects the distribution of water molecules in the AQPs water channel and it is necessary to examine the binding mode of Asn residues to water molecules in the phospholipid membrane closer to the AQPs physiological environment. 17O solid-state NMR is a powerful tool for studying the interactions between water molecules and membrane proteins in phospholipid membranes. This project is planning to use 15N selectively labeled Asn residues and 17O labeled water molecules to observe 15N-17O correlation, 15N-17O distance, and 17O quadrupolar coupling constant through DNP-enhanced solid-state NMR experiments D-HMQC, ZF-TEDOR and 3QMAS. Combining the above experimental information and quantum chemistry calculations, the way in which Asn residues in AqpZ combined with water molecules will be revealed, and the proton repulsion mechanism of AQPs in phospholipid membranes will be clarified.