Bacterial Export of Folded Proteins: Transport Mechanism of the Tat Translocon

 描述（由申请人提供）：由于蛋白质存在于细胞内的每个膜和水室中，但主要在细胞质核糖体上合成，因此蛋白质靶向和转运穿过或进入脂质膜是所有生物体的基本过程。存在许多不同类型的易位系统，其允许大蛋白质分子穿过膜而不损害膜作为离子、代谢物和大分子的渗透性屏障的作用。虽然许多蛋白质转运系统转运“线性化多肽”，但双精氨酸转运（达特）系统转运完全折叠的和 组装蛋白质而不破坏离子梯度。细菌中功能性达特系统的缺失常常导致生长缺陷和偶尔的死亡。达特机制还负责输出对人类细菌毒力重要的蛋白质。由于包括人类在内的动物不含达特机器蛋白的同源物，因此达特转运抑制剂有可能用作新型抗生素。我们的长期目标是在分子水平上破译细菌达特机器的蛋白质转运机制。在过去的研究中，已经开发了许多体外试验，使大肠杆菌达特转运机制的生物化学和生物物理学研究。最近的工作表明，达特机制催化插入的信号肽发夹到膜中的能量依赖性的方式，和信号肽的C-末端的完全易位需要一个质子动力。这些结果表明，信号肽的结合相互作用及其膜转位是直接促进成熟结构域运输的关键。然而，易位孔的结构和低聚物性质以及如何防止渗漏仍然是未解决的主要问题。易位孔的表征特别具有挑战性，因为它在不存在质子动力的情况下分解。我们将研究达特机器的结构和动力学特性，并测试我们的发夹铰链模型产生的假设。我们的具体目标是：（1）鉴定TatBC受体复合物上的信号肽结合位点，并确定TatBC寡聚体中TatBC异二聚体的排列;（2）使用实时动力学方法表征达特转运期间的转位子和货物动力学;和（3）鉴定TatA在转运期间经历的构象和环境变化。我们将使用交联和实时荧光方法，包括单分子研究，以探测结合相互作用和重要的结构中间体之间的动态转换在易位周期。这项工作将清楚地建立信号肽与达特机制相互作用的方式，以及易位孔的结构、动力学和寡聚体性质。总之，这项研究将显着推进我们的理解的达特运输机制，这反过来将提供一个健全的框架，了解细菌的生长和毒力，并为抗生素和生物技术工具的发展。

 DESCRIPTION (provided by applicant): Since proteins are found in every membrane and aqueous compartment within cells and yet are primarily synthesized on cytoplasmic ribosomes, protein targeting and transport across or into lipid membranes is a fundamental process in all organisms. Many distinct types of translocation systems exist that allow large protein molecules to cross membranes without compromising the membranes' role as a permeability barrier to ions, metabolites, and macromolecules. While many protein transport systems translocate 'linearized polypeptides', the twin-arginine translocation (Tat) system transports fully-folded and assembled proteins without collapsing ion gradients. The absence of a functional Tat system in bacteria often leads to growth defects and occasionally death. The Tat machinery is also responsible for the export of proteins important for bacterial virulence in humans. Since animals, including humans, do not contain homologues of Tat machinery proteins, inhibitors of Tat transport could potentially find use as novel antibiotics. Our long-term goal is to decipher the mechanism of protein transport by the bacterial Tat machinery at a molecular level. In past research, numerous in vitro assays that enable biochemical and biophysical investigations of the Escherichia coli Tat transport mechanism have been developed. Recent work indicates that the Tat machinery catalyzes insertion of a signal peptide hairpin into the membrane in an energy-independent manner, and that full translocation of the C-terminal end of the signal peptide requires a proton motive force. These results establish that the signal peptide's binding interactions and its membrane translocation are critical for directly promoting mature domain transport. However, the structural and oligomeric nature of the translocation pore and how leakage is prevented remain major unsolved problems. Characterization of the translocation pore is particularly challenging because it disassembles in the absence of a proton motive force. We will examine structural and dynamic properties of the Tat machinery, and test hypotheses generated by our Hairpin-Hinge Model of transport. Our Specific Aims are: (1) to identify the signal peptide binding site on the TatBC receptor complex, and determine the arrangement of TatBC heterodimers in the TatBC oligomer; (2) to characterize translocon and cargo dynamics during Tat transport using real-time kinetic approaches; and (3) to identify the conformational and environmental changes that TatA experiences during transport. We will use crosslinking and real-time fluorescence approaches, including single molecule studies, to probe binding interactions and the dynamic transitions between important structural intermediates in the translocation cycle. This work will clearly establish the manner in which signal peptides interact with the Tat machinery, and the structural, dynamic, and oligomeric properties of the translocation pore. In total, this study will significantly advance our understanding of the Tat transport mechanism, which will in turn provide a sound framework for understanding bacterial growth and virulence, and for the development of antibiotics and biotechnological tools.