Chaperone-Assisted Structure Determination of Membrane Proteins

 描述(申请人提供)：膜蛋白是一种复杂的分子机器，其功能由一系列程序化的构象转变决定。试图建立将膜蛋白动力学与它们诱导的功能联系起来的基本分子机制的努力，受到了一些看似不可逾越的技术障碍的阻碍。这些障碍中的主要原因是构象转变太短暂，不能用传统的结构生物学技术来研究。为了克服这些障碍，有人计划开发和实施一套基于噬菌体展示产生的合成抗体(Sabs)的新方法和试剂，为以前所未有的细节研究膜蛋白瞬时状态的分子性质提供新的手段。其概念是将这些SAB用作定制的结晶伴侣或单颗粒Cryo-EM应用的基准标记。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颖的噬菌体展示分选策略，可以赋予sabs特殊的性质，为研究人员提供工具来解决复杂的结构和生物问题，这些问题以前被认为过于令人望而生畏，甚至无法考虑。第二个技术进步是在充满脂质的纳米盘中进行这些选择，以更真实地概括天然的膜环境。这种环境将更好地稳定作用力诱导的所需构象状态，因此，选定的SABS将捕获和稳定真正的动态中间体，然后可以用X射线或低温EM方法进行研究。为了进一步提高这些试剂作为结晶伴侣的成功率，将使用抗体工程来改造SAB支架，以有效地增加从每个SAB结合剂中衍生的潜在伴侣的数量。为了检验和评价这些方法，将使用三类膜离子通道作为模型系统。这些系统一直拒绝使用传统方法进行结构分析，因此，将很好地衡量伴侣辅助结构确定技术的性能。

 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.