Single-molecule studies of Sec-dependent protein translocation

项目摘要 许多必需的蛋白质被插入细胞膜或分泌。因为它们是在胞浆中合成的， 这些蛋白质必须穿过脂质双层才能到达目的地并发挥功能。大多数人 结合到分泌或膜插入的蛋白质通过普遍保守的SEC易位子。 SEC途径允许从胞浆中输出的蛋白质穿过内质网 真核生物中的膜和细菌中的质膜。证券交易委员会的基本作用 许多细胞通路中的易位使得潜在的分子机制和 重任在肩。SEC系统的底物包括毒力因子和抗生素灭活酶 细菌，范围从胰岛素到动物的抗体。SEC通路中的扰动可导致 许多疾病，包括癌症和糖尿病。对易位过程的机械性理解 可以促进针对这一中枢细胞途径的药物的开发。 依赖于SEC的蛋白质转位已被广泛地用生化和结构方法研究， 描述了它的许多组成部分。然而，这一过程的动态还没有被很好地理解。在.期间 易位，转运子通道必须允许多肽在保持通透性的同时移动 离子和其他溶质的屏障。通道如何与多肽底物相互作用来实现这些 看似相互冲突的要求尚不清楚。另一个尚未回答的关键问题是如何 与转位子有关的分子机器将化学能转化为机械功。 这为移位提供了动力。关于SEC易位的许多重要悬而未决的问题 如果可以实时跟踪蛋白质通过通道的通道，则可以对系统进行应答 具有较高的空间和时间分辨率，但目前尚不具备这种能力。 能够观察和操纵单个大分子的单分子方法具有 对生物机制提供了前所未有的见解。我建议调查SEC的机制- 用光学镊子进行依赖的蛋白质转位。这种方法使我们能够解开 潜在的SEC依赖的蛋白质易位。我们的研究将产生新的和令人兴奋的见解 转位机制用来将蛋白质运出细胞质的机制。

Project Summary Many essential proteins are inserted into membranes or secreted. Because they are synthesized in the cytosol, these proteins must cross a lipid bilayer to reach their destination and become functional. The majority of proteins bound for secretion or membrane insertion transits through the universally conserved Sec translocon. The Sec pathway allows proteins destined for export from the cytosol to cross the endoplasmic reticulum membrane in eukaryotes and the plasma membrane in bacteria. The essential role of Sec-dependent translocation in many cellular pathways makes elucidation of the underlying molecular mechanisms an important task. Substrates of the Sec system include virulence factors and antibiotic-inactivating enzymes in bacteria, and range from insulin to antibodies in animals. Perturbations in the Sec-pathway can lead to numerous diseases, including cancer and diabetes. A mechanistic understanding of the translocation process can fuel the development of drugs that target this central cellular pathway. Sec-dependent protein translocation has been studied extensively with biochemical and structural approaches, characterizing many of its components. However, the dynamics of the process are not well understood. During translocation, the translocon channel must allow the polypeptide to move while maintaining a permeability barrier for ions and other solutes. How the channel interacts with polypeptide substrates to achieve these seemingly conflicting requirements is not known. Another key question that has remained unanswered is how the molecular machines that associate with the translocon convert chemical energy into the mechanical work that powers translocation. Many of the important outstanding questions concerning the Sec translocation system could be answered if it were possible to follow the passage of a protein through the channel in real-time with high spatial and temporal resolution, but this capability is not presently available. Single-molecule approaches, enabling observation and manipulation of individual macromolecules, have provided unprecedented insights into biological mechanisms. I propose to investigate the mechanisms of Sec- dependent protein translocation with optical tweezers. This approach enables us to unravel the mechanisms underlying Sec-dependent protein translocation. Our studies will yield new and exciting insights into the mechanisms employed by the translocation machinery to transport proteins out of the cytosol.