前期研究表明，UmpAB代表一类新型双组分Na+(Li+, K+)/H+逆向转运蛋白。然而，其行使功能必须依赖“异源二聚体”形成的原因仍然未知。作为新型Na+/H+逆向转运蛋白，其分子转运机制也有待分析。因此，项目拟采取拓扑学分析及PhoA显色/活性实验，明确UmpAB双拓扑结构特征；进而采用重叠PCR构建UmpAB中3个独特亲水区的缺失或互换突变体，结合功能互补、活性分析及Western blot等实验，鉴定以上亲水区对其双拓扑结构的功能重要性；在此基础上，通过定点突变，结合功能互补、活性分析及Western blot等，佐以FRET、Co-IP、ITC、质子释放等实验，明确影响二聚体形成或结合质子或Na+的关键残基；最终揭示UmpAB以异源二聚体的形式行使功能的原因及其分子转运机制，以增进对高度同源的异源二聚体的结构与功能关系的认识与理解，并为其分子改造及在耐盐碱方面应用提供技术指导。

Preliminary experiments reveal that UmpAB should represent a class of novel two-component Na+(Li+, K+)/H+ antiporters. However, it's so intriguing to discover why UmpAB must form a hetero-dimer to exhibit high Na+(Li+, K+)/H+ antiport activity. As a novel Na+/H+ antiporter, the molecular mechanim of UmpAB for Na+(Li+, K+)/H+ antiport remains to be further clarified. In this project, we plan to carry out a topological prediction via a web-based software and a coloration and activity assay using PhoA fusions with UmpA or UmpB to determine its characteristics of dual topology structure. Also, we plan to construct the deletion or substitution variants of three unique hydrophilic regions to identify whether a dual topology structure may play a vital role in the function of UmpAB as a Na+(Li+, K+)/H+ antiporter. On the basis of these results, we plan to first screen the important residues for Na+(Li+, K+)/H+ antiport activity of UmpAB via site-directed mutagenesis combined with functional complementation tests, Na+(Li+, K+)/H+ antiport assay and western blot. And then we plan to find out the key residues for the formation of hetero-dimer by using fluorescence resonance energy transfer (FRET) and Co-immunoprecipitation (Co-IP). Also, we plan to clarify Na+-binding or H+-binding sites of UmpAB by using purified UmpAB and their variants that can lead to the loss of Na+(Li+, K+)/H+ antiport activity of UmpAB via isothermal titration calorimetry (ITC) and H+ release experiments. Based on the above experiments, we will finally explain why UmpAB must form a hetero-dimer to exhibit high Na+(Li+, K+)/H+ antiport activity and how this transporter can function as Na+(Li+, K+)/H+ antiporter. The related conclusions from this project will positively contribute to the knowledge of the structure-function of highly homologous hetero-dimers, and point out an accurate direction for the molecular-level modification of UmpAB and its application in bacterial halo-alkaline tolerance.