新型Na+/H+逆向转运蛋白WEHH的结构-功能关系研究

Na+/H+逆向转运蛋白对细菌耐盐碱至关重要。该类蛋白常以12个跨膜区形成经典的NhaA折叠行使功能。前期研究证实，功能未知的膜蛋白WEHH代表一类新型Na+/H+逆向转运蛋白。然而WEHH全长仅103个残基且可能只具有2-3个跨膜区。如此小的蛋白基于何种结构-功能关系行使Na+/H+逆向转运活性有待分析。我们推测WEHH可能形成寡聚体以实现类似的NhaA折叠。因此，本项目拟通过PhoA融合表达鉴定WEHH的跨膜区数量；进而通过FRET、Co-IP及BN-PAGE等实验，鉴定WEHH是否形成寡聚体及以几聚体形式存在；在此基础上，通过定点突变，结合功能互补和活性鉴定、质子释放及ITC等，鉴定WEHH寡聚体形成及质子和Na+结合的重要残基，以最终揭示WEHH的结构-功能关系。项目的开展可增进Na+/H+逆向转运蛋白分子转运机制的认识和理解，且对NhaA折叠结构的进化机制同样具有重要启示作用。

Na+/H+ antiporters play a vital role in the halo-alkaline tolerance of bacteria. These antiporters are usually composed of twelve transmembrane helices (TMHs), which are used to form a classical structural fold designated as NhaA fold. Preliminary experiments reveal that an unknown membrane protein designated as WEHH should represent a novel class of Na+/H+ antiporters. However, WEHH is only 103 amino acid residues in full length and may consist of two - three TMHs. We speculate that WEHH may assemble into a homo-oligomer to form a similar structural fold to that of NhaA fold. The structure-function relationship of such small antiporter for Na+/H+ antiport activity remains to be analyzed. In this project, we plan to identify the number of transmembrane regions of WEHH by using PhoA fusions with WEHH via an alkaline phosphatase activity assay. Also, we plan to analyze whether and how it forms a homo-oligomer by using Förster Resonance Energy Transfer (FRET), Co-Immunoprecipitation (Co-IP) and BN-PAGE combined with immunoblotting. On this basis, we plan to carry out site-directed mutagenesis of potential functionally important amino acid residues for H+ and Na+ binding and the ones for the oligomeration of WEHH, and functional complementation tests, and assays for Na+/H+ antiport activity, and assays for H+ release, and isothermal titration calorimetry (ITC) experiments. Finally, we will discover the structure-function relationship of WEHH. The expected findings will be helpful to not only contribute positively to the knowledge of molecular transporting mechanisms of Na+/H+ antiporters, but also provide an important implication for the evolutionary mechanism of the NhaA fold.