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

呼吸复合体I由13个不同的亚基和10个辅因子组成，其组装和详细功能尚不清楚。在这个项目中，我们将结晶来自高温嗜热性的Aquifex aeolicus的络合物和它的部分。我们在大肠杆菌中检测到几种形式的部分组装的复合体，可能与染色体缺失突变体中的伴侣相关。变异体的电子转移反应和质子转移将被量化，以深入了解络合物的机理。此外，将通过免疫学方法对突变体进行筛选，以确定是否存在酶失活的变异体。变异体与可能的伴侣之间的相互作用将通过定点交联进行分析，并在体内使用荧光显微镜进行定量。氧化磷酸化复合体在不同的膜结构域中的可能定位将通过活细胞成像进行研究。为了这个目的，已经用荧光蛋白标记了复合体I、复合体II、BD和BO氧化酶以及ATP合成酶。将分析不同膜结构域内和膜结构域之间的络合物的动力学。通过FRAP和FRET实验，将在含有几个不同颜色的络合物的菌株中确定络合物之间的相互作用。考虑到脂质的作用，我们计划在体内和体外检测可能的超复合体的形成。

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.