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

两种化学驱动的钠转运蛋白复合物的结构研究

• 批准号: 277935450
• 负责人: Dr. Stella Vitt
• 金额: --
• 依托单位: Fachbereich 17: Biologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/277935450?language=en
• 关键词: Structural; studies; two; chemically; driven

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.

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，这与呼吸作用中氧化磷酸化的典型途径完全不同。在此背景下，我提出了一个由两类跨膜多亚单位蛋白质复合体的结构和功能研究组成的项目。两者都起着钠离子泵的作用，是几个发酵途径节能的关键部件。Na+-转位生物素脱羧酶(Na+-BDC)利用厌氧细菌中的代谢物--谷氨酰辅酶A等芳酮酸类化合物的放热脱羧化反应。H+/Na+转位NAD+-铁氧还蛋白还原酶(RNF)通过黄素基电子分叉产生的能量丰富的铁还蛋白还原NAD+。首先，我计划纯化发酵酸氨基球菌的谷氨酰辅酶A脱羧酶和四异形梭菌的RNF，并进行单颗粒冷冻电子显微镜(EM)分析，以获得质量信息和低分辨率结构。同时，每个复合体的可溶亚基将被异源产生，以产生可以适应低分辨率地图的高分辨率结构。在通过洗涤剂类型和脂类含量的变化进一步优化了完整络合物的均一性后，还用-in表面和-in介观方法计划了它们的结晶。这个项目具有挑战性，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，它们的异源/同源生产可能是必要的。此外，我还打算证明纯RNF的Na+转运活性。