Tat translocon assembly states and structures

双精氨酸易位（达特）系统用于运输折叠的、通常含有辅因子的蛋白质穿过细菌、古细菌、质体以及一些线粒体中的能量转导膜。依赖Tat转运的蛋白质是由含有表肽“双精氨酸基序”的转运介导的N-末端信号肽合成的。达特途径对于光合和许多呼吸氧化还原途径是必需的，并且由于其涉及许多病原体的毒力，包括高度重要的物种，如结核分枝杆菌或铜绿假单胞菌，它也是特别感兴趣的。当前达特研究的一个关键问题是，达特系统如何机械地使直径甚至大于膜厚度的蛋白质能够转运。达特转座子由至少两种不同蛋白质的多个拷贝形成，所述两种不同蛋白质为TatA/B家族的一个成员和TatC。在深入研究的模式生物大肠杆菌中，完全活性的转座子可以由三种组分TatA、TatB和TatC形成。我们分析了活性TatABC translocons的组装，并能够区分三个无底物的达特复合物（TC 1，TC 2，TC 3）和两个底物相关的复合物（TC 1 S和TC 2S），通过蓝色的非变性聚丙烯酰胺凝胶电泳（BN PAGE）。基于这些和以前的数据，我们开发了一个工作模型的易位组装步骤和随之而来的易位事件。在这个项目中，我们的目标是区分这些不同的达特复合物的位点特异性诱变结合位点特异性交联。我们已经确定了几个突变，丰富的达特复合物的某些类型，这些变化的复杂的分布，现在允许识别复杂的特定的交联。该项目的第一部分侧重于这些方面，并将交叉链接分析扩展到更多的位置，这将导致在不同的条件下或生长阶段的translocon状态的变化的评估。该项目的第二部分侧重于纯化的达特复合物的生化和结构分析。对于允许分析底物结合参数、亚基化学计量或冷冻EM结构测定的规模的纯化，将通过不同的方法稳定达特复合物，包括使用交联以及新的不依赖于去污剂的增溶方法。总之，这个项目的两个部分将有助于理解完全折叠的蛋白质穿过生物膜的迷人运输机制。

The twin-arginine translocation (Tat) system serves to transport folded, often cofactor-containing proteins across energy-transducing membranes in bacteria, archaea, plastids and also some mitochondria. Tat-dependently transported proteins are synthesized with a transport-mediating N-terminal signal peptide that contains the eponymous “twin-arginine motif”. The Tat pathway is essential for photosynthetic and many respiratory redox pathways, and it is also of special interest due to its involvement in virulence of many pathogens, including highly important species such as Mycobacterium tuberculosis or Pseudomonas aeruginosa. A key question of current Tat research is, how the Tat system mechanistically enables transport of proteins that can be even larger in diameter than the membrane thickness. Tat translocons are formed by multiple copies of minimally two distinct proteins, one member of the TatA/B family and a TatC. In the intensively studied model organism Escherichia coli, fully active translocons can be formed by the three components TatA, TatB, and TatC. We analyzed the assembly of active TatABC translocons and were able to distinguish three substrate-free Tat complexes (TC1, TC2, TC3) and two substrate associated complexes (TC1S and TC2S) by blue-native polyacrylamide gel electrophoresis (BN PAGE). Based on these and previous data, we developed a working model for translocon assembly steps and the concomitant translocation events. In this project, we aim to differentiate these distinct Tat complexes by site specific-mutagenesis in combination with site-specific cross-linking. We have already identified several mutations that enrich Tat complexes of certain types, and these changes of the complex distributions now permit the identification of complex-specific cross-links. The first part of the project focuses on these aspects and will extend the cross-link analyses to a larger number of positions, which will result in the assessment of changes of the translocon status under distinct conditions or growth phases. The second part of the project focuses on the biochemical and structural analyses of purified Tat complexes. For purifications in scales that permit analyses of substrate binding parameters, subunit stoichiometry or cryo-EM structure determination, Tat complexes will be stabilized by different approaches, including the use of cross-linking as well as novel detergent-independent solubilization methods. Together, both parts of this project will help to understand the still enigmatic mechanism for the fascinating transport of fully folded proteins across biological membranes.