Interaction of ECF and ABC transporter modules studied by time resolved fluorescence microscopy and spectroscopy

通过时间分辨荧光显微镜和光谱学研究 ECF 和 ABC 转运蛋白模块的相互作用

• 批准号: 137912872
• 负责人: Professor Dr. Andreas Herrmann
• 金额: --
• 依托单位: Institut für Chemie und Biochemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2009
• 资助国家: 德国
• 起止时间: 2008-12-31 至 2013-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/137912872?language=en
• 关键词: Interaction; ECF; ABC; transporter; modules

The organisation and structure of ECF ("energy-coupling factor") transporters (BioMNY, EcfAA`T) and the histidine ABC transporter (HisJ/LAO-HisQMP2) during the catalytic cycle of the transporters will be investigated by time resolved fluorescence microscopy and spectroscopy. Various techniques including Fluorescence Lifetime Imaging, Förster-Resonance-Energy-Transfer, Fluorescence (Cross) Correlation spectroscopy and Fluorescence Recovery after Photobleaching will be employed to study the interaction of the various subunits and modules of transporters and their conformational dynamics. Transporter subunits will be labelled either by fluorescent proteins or by reactive fluorophores. By tagging of transporter subunits with fluorescent proteins, measurements to characterize subunit interactions can be performed on living cells (E. coli) and on detergent solubilized, liposome (Giant Unilamellar Vesicles) and NanoDisc reconstituted modules. NanoDiscs will be used to characterize the organisation of transporter at single molecule level by fluorescence bleaching. Biochemical assays provided by T. Eitinger and E. Schneider (see respective proposals) will be used to probe for function of fluorescently labelled transporters. Our studies complement those by Electron Spin Resonance (see project proposals of T. Eitinger, E. Schneider and H.J. Steinhoff)

将通过时间分辨荧光显微镜和光谱法研究ECF（“能量偶联因子”）转运蛋白（BioMNY，EcfAA`T）和组氨酸ABC转运蛋白（HisJ/LAO-HisQMP 2）在转运蛋白催化循环期间的组织和结构。各种技术，包括荧光寿命成像，Förster共振能量转移，荧光（交叉）相关光谱和光漂白后的荧光恢复将被用来研究转运蛋白的各种亚基和模块的相互作用及其构象动力学。转运蛋白亚基将被荧光蛋白或反应性荧光团标记。通过用荧光蛋白标记转运蛋白亚基，可以在活细胞上进行表征亚基相互作用的测量（E。coli）和去污剂溶解的脂质体（巨单层囊泡）和NanoDisc重构模块上。NanoDiscs将用于通过荧光漂白在单分子水平表征转运蛋白的组织。生物化学测定由T. Eitinger和E. Schneider（见相应提案）将用于探测荧光标记转运蛋白的功能。我们的研究补充了电子自旋共振（见T。Eitinger，E. Schneider和H.J. Steinhoff）