Structural studies on two chemically driven Na+-translocating protein complexes

进化发展了令人着迷的酶机制，将化学能转化为电化学离子梯度，这与呼吸过程中氧化磷酸化的典型途径完全不同。在此背景下，我提出了一个项目，包括两类跨膜多亚基蛋白复合物的结构和功能研究。两者都作为钠离子泵，是几个发酵途径中能量守恒的关键组成部分。含Na+易位生物素脱羧酶（Na+-Bdc）利用ß-酮酸样化合物（如谷氨酰基辅酶a）的放热脱羧反应，这是厌氧细菌的代谢产物。H+/Na+易位的NAD+-铁氧还蛋白还原酶（Rnf）通过黄素电子分岔产生的富含能量的铁氧还蛋白来还原NAD+。我计划首先纯化发酵酸胺球菌的谷氨酰辅酶a脱羧酶和破伤风梭菌的Rnf，并进行单粒子冷冻电镜（EM）分析，获得高质量的信息和低分辨率的结构。同时，每个复合物的可溶亚基将被异质地产生，以产生高分辨率的结构，这些结构可以适合于低分辨率的地图。在通过洗涤剂类型和脂质含量的变化进一步优化了完整配合物的均匀性之后，他们的结晶也计划与-表面和-介观方法。该项目具有挑战性，但最有价值的是详细了解这些化学驱动的Na+泵的作用模式。在后期，可能需要它们的异源/同源生产。此外，我打算证明纯种破伤风球菌Rnf的Na+转运活性。

Evolution developed fascinating enzymatic machineries to transform chemical energy into an electrochemical ion gradient that completely differ from those of the canonical pathway of oxidative phosphorylation in respiration. In this context I propose a project that consists of structural and functional studies on two classes of membrane-spanning multisubunit protein complexes. Both act as Na+ pumps and are key components in the energy conservation of several fermenting pathways. Na+-translocating biotin containing decarboxylases (Na+-Bdc) exploit the exothermic decarboxylation reaction of ß-ketoacid-like compounds such as glutaconyl-CoA, which are metabolites in anaerobic bacteria. The H+/Na+-translocating NAD+-ferredoxin reductase (Rnf) reduces NAD+ by the energy-rich ferredoxin produced by flavin-based electron bifurcation. I plan, at first, to purify glutaconyl-CoA decarboxylase of Acidoaminococcus fermentans and Rnf of Clostridium tetanomorphum and perform a single-particle cryo-electron microscopy (EM) analysis to obtain quality information and a low-resolution structure. In parallel, soluble subunits of each complex will be heterologously produced to generate high-resolution structures that can be fitted into the low-resolution maps. After further optimizing the homogeneity of the complete complexes by detergent type and lipid content variations, their crystallization is also planned with the -in surfo- and -in meso- methods. This project is challenging but most valuable to understand the mode of action for these chemically driven Na+ pumps, in detail. In later stage their heterologous/homologous production might be necessary. In addition, I intend to proof the Na+-translocating activity of pure Rnf of C. tetanomorphum.