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

• Applicant: Dr. Stella Vitt
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms ; Structural Biology
• Term: from 2015 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 277935450

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.

• DFG Programme: Research Grants