Assembly of E. coli complex I and its interaction with other proteins

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The respiratory complex I is composed of 13 different subunits and 10 cofactors, whose assembly and detailed function are still unclear. In this project, we will crystallize the complex and parts of it from the hyperthermophile Aquifex aeolicus. We have detected several forms of a partially assembled complex possibly associated with chaperones in chromosomal deletion mutants in Escherichia coli. The electron transfer reaction and proton translocation of the variants will be quantified to gain insight into the mechanism of the complex. In addition, the mutants will be screened for the presence of enzymatically inactive variants by immunological methods. The interaction of the variants with putative chaperones will be analyzed by site-directed crosslinking and quantified in vivo using fluorescence microscopy. The possible localization of the complexes of oxidative phosphorylation in distinct membrane domains will be investigated by live cell imaging. For this purpose complex I, complex II, the bd and the bo oxidases, and the ATP-Synthase have been labeled with fluorescent proteins. The dynamics of the complexes within and between distinct membrane domains will be analyzed. The interactions between the complexes will be determined in strains containing several complexes decorated with different colors by FRAP and FRET experiments. We plan to detect the possible formation of supercomplexes in vivo and in vitro considering the role of lipids.