'Structure and function of the R-body, a piston-like nanomachine

项目摘要 R-小体是草履虫菌株的细菌共生体自组装的蛋白质聚合物，这种共生体可以 赋予宿主杀死相互竞争的菌株的能力。紧密缠绕的亚微米大小的R-Body经历了 大量的pH诱导构象变化成几十微米长的细长棒，产生作用力 足以破坏生物膜。纯化的重组R小体可以经历多次连续的 几轮活塞式的伸展和收缩只利用了pH值变化产生的化学能。这些健壮的 纳米机器在生物技术和治疗应用方面具有巨大的潜力。他们有能力 充当灵敏的开关并在生物结构上做功可以适应许多功能-它们 例如，已经提出作为吞噬体递送生物活性分子的系统，其方式 会模仿它们的生物学功能。调整R-Body行为的能力最近被一种 一组点突变，可以改变延伸的触发pH值。然而，缺乏详细的分子 了解它们的组装和pH敏感的活塞状延伸的机制是主要的 为新的应用开发R-Body的障碍。这项提议旨在确定分子 使用混合结构方法的R形体的结构，并使用 光学捕获力测量。可视化盘绕的R形体的三维结构 扩展状态将允许我们定义它们活塞式行为的分子机制，为 调整结构以适应新功能的基础工作。

Project Summary R-bodies are self-assembling protein polymers from bacterial ensymbionts of 'killer' Paramecium strains that confer to the host the ability to kill competing strains. The tightly coiled sub-micron sized R-body undergoes a massive pH induced conformational change into elongated rods tens of microns long, generating forces sufficient to rupture biological membranes. Purified recombinant R-bodies can undergo many successive rounds of piston-like extension and contraction using only the chemical energy from pH change. These robust nanomachines have enormous potential for biotechnological and therapeutic applications. Their ability to behave as sensitive switches and to do work on biological structures can be adapted for many functions - they have, for example, been proposed as systems for phagosomal delivery of bioactive molecules in a way that would mimic their biological function. The ability to tune R-body behavior was recently demonstrated by a panel of point mutants that alter the triggering pH for extension. However, the lack of detailed molecular understanding of their assembly and the mechanism of the pH-sensitive piston-like extension are major roadblocks to developing R-bodies for novel applications. This proposal aims to determine the molecular architecture of R-bodies using a hybrid structural approach, and to measure their biophysical properties using optical trapping force measurements. Visualizing the three-dimensional structure of R-bodies in coiled and extended states will allow us to define the molecular mechanism of their piston-like behavior, laying the groundwork for tuning the structures to new functions.