Structural and Functional Analysis of Bacterial Formate Channels

甲酸盐是细菌的主要代谢物，在各种呼吸途径中作为电子供体起着关键作用。甲酸盐在生物膜上的转运是由甲酸通道FocA介导的，它是膜转运蛋白FNT家族的成员。我们对鼠伤寒沙门菌蛋白在低pH条件下的三维结构进行了研究，结果显示，n端发生了一系列协调一致的构象变化，形成了一个功能转换的基础，即在高外部pH条件下通过促进扩散输出甲酸阴离子，在低于6.8的pH条件下输入二级活性甲酸/H+。这对应于一种代谢转变，从丙酮酸：甲酸裂解酶（PFL）产生的细胞质甲酸和周围质呼吸性甲酸氧化到甲酸：氢裂解酶复合物（FHL）产生的细胞质甲酸歧化。我们建议继续并扩展我们在FocA上的工作，包括甲酸通道的电生理表征及其对脂质双分子层中质子动力的依赖。我们将通过位点定向诱变进一步分析FocA的pH感应机制，并研究该蛋白的亚细胞定位及其与生理伙伴（如PFL或FHL复合物）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.