Dynamics of subunit interactions in ECF transporters

ECF 转运蛋白中亚基相互作用的动力学

• 批准号: 137770702
• 负责人: Professor Dr. Thomas Eitinger
• 金额: --
• 依托单位: Arbeitsgruppe Mikrobiologie
• 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 2016-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/137770702?language=en
• 关键词: Dynamics; subunit; interactions; ECF; transporters

Energy-coupling factor (ECF)" transporters are composed of a substrate-specific (S) and a moderately conserved (T) integral membrane protein and of ABC ATPases. In contrast to canonocal ABC importers, ECF systems do not rely on extracytoplasmic solute-binding proteins. The oligomeric composition of A-, T- and S-units, the exact roles of the S- and T-components and the molecular mechanisms that couple dynamic interactions during ATP hydrolysis to substrate translocation are controversial or unknown, respectively. Crystal structure analysis identified three different S-units as substrate-loaded monomers. In vivo investigations, site-directed mutagenesis and crosslinking experiments, on the other hand, favor oligomeric states of the S- and potentially the T-units as the functional in vivo states. The focus of the present application is on biotin transporters (BioYMN). Our previous results favor a BioY dimer as the functional S-unit that has transport (in addition to substrate-binding) activity in its solitary state. An appropriate E. coli reporter strain will be used to clarify eventually the controversially discussed issue of BioY's transport function using a set of BioY homologs. The oligomeric states of the T-unit BioN and of BioY will be analyzed by fluorescence-spectroscopical and –imaging techniques using live cells as well as purified transporter complexes reconstituted in nanodiscs. EPR techniques will be applied to spin-labeled complexes in nanodiscs as a complementary approach. The dynamics of subunit interactions during ATP hydrolysis will be investigated by kinetic analysis, pull-down and EPR experiments. An ongoing structural biology approach in external collaboration aims at solving the 3D structure of an ECF holotransporter as a major step towards an understanding the subunit organization.

能量偶联因子（ECF）转运蛋白由底物特异性（S）和中度保守性（T）整合膜蛋白和ABC ATP酶组成。与典型的ABC进口商相反，ECF系统不依赖于胞质外溶质结合蛋白。A-，T-和S-单元的寡聚体组成，S-和T-组件的确切作用和耦合ATP水解过程中的动态相互作用的分子机制，底物易位是有争议的或未知的，分别。晶体结构分析确定了三种不同的S-单元作为底物负载单体。另一方面，体内研究、定点诱变和交联实验有利于S-和潜在的T-单元的寡聚状态作为体内功能状态。本申请的焦点是生物素转运蛋白（BioYMN）。我们以前的研究结果有利于BioY二聚体作为功能性S-单元，在其孤立状态下具有运输（除了底物结合）活性。一个合适的E.大肠杆菌报告菌株将被用来澄清最终有争议的讨论问题的BioY的运输功能使用一套BioY同源物。T-单元BioN和BioY的寡聚状态将通过荧光光谱和成像技术使用活细胞以及在纳米盘中重构的纯化的转运蛋白复合物进行分析。EPR技术将被应用于自旋标记的复合物在纳米盘作为一种补充的方法。ATP水解过程中亚基相互作用的动力学将通过动力学分析，下拉和EPR实验进行研究。一个正在进行的结构生物学方法在外部合作的目的是解决三维结构的ECF holotransporter作为一个重要的一步，了解亚基组织。