Chaperone-Assisted Structure Determination of Membrane Proteins

 描述（由申请人提供）：膜蛋白是复杂的分子机器，其功能由一组程序化构象转换控制。试图建立基本的分子机制，连接膜蛋白的动态功能，他们诱导已被挫败了一些看似不可逾越的技术障碍。这些障碍中最主要的是构象转变太短暂，无法使用传统的结构生物学技术进行研究。为了克服这些障碍，提出了一个计划，以开发和实施一套新的方法和试剂的基础上噬菌体展示产生的合成抗体（sAB），提供新的手段来研究膜蛋白的瞬态的分子特性在前所未有的细节。该概念是使用这些sAB作为定制的结晶伴侣或基准标记用于单颗粒Cryo-EM应用。为了推动这些发展，将结合两种强大的技术方法。第一项技术是一套新的噬菌体展示分选策略，可以赋予sAB特殊的性质，为研究人员提供解决复杂结构和生物学问题的工具，这些问题以前被认为过于艰巨，甚至无法考虑。第二个技术进步是在脂质填充的纳米盘中进行这些选择，这些纳米盘更忠实地再现了天然膜环境。这种环境将更好地稳定由力诱导的所需构象状态，因此，所选择的sAB将捕获并稳定真正的动态中间体，然后可以通过X射线或Cryo-EM方法进行研究。为了进一步提高这些试剂作为结晶分子伴侣的成功率，将采用抗体工程改造来重塑sAB支架，以有效地使可衍生自每种sAB结合剂的潜在分子伴侣的数量倍增。为了测试和评估的方法，三类膜离子通道将被用作模型系统。这些系统已经被使用传统的方法进行结构分析，因此，将提供一个很好的措施的伴侣辅助结构测定技术的性能。

 DESCRIPTION (provided by applicant): Membrane proteins are complex molecular machines whose functions are governed by sets of programed conformational transitions. Attempts to establish the fundamental molecular mechanisms that link membrane protein dynamics to functions they induce have been thwarted by a number of seemingly insurmountable technical barriers. Principal among these barriers is that the conformational transitions are too transient t be studied using traditional structural biology techniques. To overcome these barriers, a plan is proposed to develop and implement a set of novel methodologies and reagents based on phage display generated synthetic antibodies (sABs) that provide new means to study the molecular properties of transient states of membrane proteins at unprecedented detail. The concept is to use these sABs as customized crystallization chaperones or fiducial marks for single particle Cryo-EM applications. To advance these developments two powerful technology approaches will be combined. The first technology is a set of novel phage display sorting strategies that can endow the sABs with special properties providing investigators the tools to tackle complex structural and biological problems that were previously thought to be too daunting to even contemplate. The second technology advance is to perform these selections in lipid-filled nanodiscs that more faithfully recapitulate the native membrane environment. This environment will better stabilize desired conformational states induced by forces and as a consequence the selected sABs will capture and stabilize true dynamic intermediates that can be then studied by X-ray or Cryo-EM methods. To further increase success rates of these reagents as crystallization chaperones, antibody engineering will be employed to remodel the sAB scaffolds to effectively multiple the number of potential chaperones that can be derived from each sAB binder. To test and evaluate the methodologies, three classes of membrane ion-channels will be used as model systems. These systems have been recalcitrant to structural analysis using traditional approaches and thus, will provide a good measure of the performance of the chaperone-assisted structure determination technologies.