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

项目摘要 R体是来自“杀手”草履虫菌株的细菌共生体的自组装蛋白质聚合物， 赋予宿主杀死竞争菌株的能力。紧密盘绕的亚微米大小的R体经历了 巨大的pH值引起构象变化，变成几十微米长的细长杆，产生力 足以使生物膜破裂纯化的重组R体可以经历许多连续的 一轮又一轮的活塞式伸展和收缩，仅仅利用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。的能力 作为敏感的开关，并在生物结构上工作，可以适应许多功能-它们 已经被提出作为吞噬体递送生物活性分子的系统， 会模仿它们的生物功能。调节R体行为的能力最近被一个 一组改变延伸的触发pH的点突变体。然而，缺乏详细的分子 了解它们的组装和pH敏感活塞样延伸的机制是主要的 为新的应用开发R体的障碍。该建议旨在确定分子 结构的R-体，并测量其生物物理特性， 光学捕获力测量。在螺旋和螺旋中可视化R体的三维结构 扩展状态将使我们能够定义其活塞式行为的分子机制，奠定了 为调整结构以适应新功能奠定基础。

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.