Structural and Functional Analysis of Bacterial Formate Channels

甲酸盐是细菌的主要代谢物，在各种呼吸途径中起着电子供体的重要作用。甲酸盐在生物膜上的转运是由FNT家族膜转运蛋白家族中的甲酸盐通道FocA介导的。我们在低pH下对鼠伤寒沙门氏菌蛋白质的三维结构显示了一组协调和合作的N-末端构象变化，这些变化形成了功能转换的基础，在高外部pH下通过促进扩散从甲酸盐负离子输出到pH低于6.8的次要活性甲酸盐/H+输入。这对应于由丙酮酸：甲酸裂解酶(PFL)和周质呼吸甲酸盐氧化产生的细胞质甲酸盐在甲酸：氢裂解酶复合体(FHL)处的代谢转变。我们建议继续和扩展我们在FOCA方面的工作，以包括甲酸盐通道的电生理特征及其对脂质双层中质子动力的依赖。我们将通过定点突变进一步分析FocA的pH敏感机制，并研究该蛋白的亚细胞定位及其与PFL或FHL复合体等生理伙伴的相互作用。

Formate constitutes a major metabolite in bacteria and plays a key role as electron donor in various respiratory pathways. The transport of formate across biological membranes is mediated by the formate channel FocA, a member of the FNT family of membrane transporters. Our three-dimensional structure of the protein from Salmonella typhimurium at low pH showed a concerted and cooperative set of conformational changes of the N-terminus that form the basis for a functional switch from the export of the formate anion by facilitated diffusion at high external pH to secondary active formate/H+ import at a pH below 6.8. This corresponds to a metabolic shift from cytoplasmic formate production by pyruvate:formate lyase (PFL) and periplasmic, respiratory formate oxidation to cytoplasmic formate disproportionation at the formate:hydrogen lyase complex (FHL). We propose to continue and extend our work on FocA to include an electrophysiological characterization of the formate channel and its dependency on the proton motive force in lipid bilayers. We will further analyse the mechanism of pH sensing in FocA by site-directed mutagenesis and investigate the subcellular localization of the protein and its interaction with physiological partners such as PFL or the FHL complex.