多家族离子共转运蛋白结构模型构建及分子转运机制研究

离子共转运蛋白能够利用Na+、H+等实现逆浓度梯度转运小分子物质，在生物体中承担着重要的生理功能。近年来作为各种疾病治疗重要的药物靶点而成为研究热点。但由于膜蛋白表达纯化及结晶等技术困难，目前仅有少数的转运蛋白获得晶体结构。我们发现来源于多个家族的相关蛋白均具有类似NhaA的折叠方式，因此拟通过已知蛋白结构和进化保守性分析建立基于蛋白结构核心功能区域和底物结合位点的筛选标准。获得符合筛选标准的蛋白序列并选取CPA、IT和BART超家族的NhaP、LctP和ArsB蛋白进行实验验证。首先选取核心功能区域保守位点进行突变，对突变体进行生理功能验证和动力学参数变化分析；构建底物结合关键位点突变体蛋白并表达纯化，通过FRET、Cross-linking水解等技术研究突变体蛋白的构象变化。最终建立准确的筛选标准和涵盖较广的结构模型，为相关转运蛋白的分子转运机制和药物设计提供理论依据。

Ion cotransporters can use Na+, H+ to mediate uphill cotransport of small molecules, and thus play important physiological functions in the organisms. Being critical targets for various diseases treatment, these transporters become a research hot spot in recent years. However, due to the technical difficulties of purification and crystallization in membrane proteins, only very few transporters had been crystalized. Based on the previous studies, we found that many proteins from multiple families possess the NhaA-like folding patterns. Therefore, we will establish screening criteria based on core functional regions and substrate binding sites, by comparing available structures and evolutionary conservation analysis. Three proteins of NhaP, LctP, and ArsB, from CPA, IT and BART superfamilies, will be used for testing the screening criteria. Some mutants involving in the core region will be constructed for physiological function verification and kinetic parameter analysis. Furthermore, the predicated sites for substrate binding will be expressed and purified, and further analyzed by FRET and Cross-linking analysis for their conformational changes. Finally, an accurate screening criteria, as well as a general structural model suitable for many sequences, will be constructed. This will provide a theoretical basis for elucidating the molecular transport mechanism of relevant transporters.