Chaperone-Assisted Structure Determination of Membrane Proteins

摘要： 膜蛋白是一种复杂的分子机器，其功能由一系列的 程序化的构象转变试图建立基本的分子机制， 膜蛋白的结构和动力学功能，他们诱导已被挫败的一些 看似不可逾越的技术壁垒这些障碍中的主要障碍是， 太过短暂，无法用传统的结构生物学技术进行研究。为了克服这些障碍，我们 已经开发并实施了一套基于噬菌体展示产生的新方法和试剂， 合成抗体（sAB）。自定义噬菌体展示选择策略能够产生赋予 具有特殊的性质，例如构象和区域特异性。这些试剂已被用于研究 膜蛋白质的瞬时状态的分子特性在前所未有的细节。虽然sAB具有 证明了作为结晶分子伴侣的功效，它们在冷冻EM中作为强大的基准标记的用途，增加了50 kDa的粒子和他们的能力，陷阱构象状态，是特别有影响力的研究， 构象转变和功能。这对于较小的膜蛋白（< 50 kDa）特别相关， 其包括离子通道转运蛋白和受体。这些构成了最大的一类生物医学相关的 靶向系统，但不容易结晶，并且对于冷冻-EM分析来说太小。充分发挥两国 当前的技术平台，我们建议设计和部署一套高阶sAB结构， 用于增加靶膜蛋白的大小、刚性，以及在某些情况下增加靶膜蛋白的对称性。这些 基于sAB的实体将被设计成预制的组装模块。他们的目标是 已经引入膜蛋白中的特异性表位，因此可以普遍使用 无论它们应用于哪个系统。这种方法的力量在于，这些“通用”sAB可以 以“即插即用”的方式添加到感兴趣的分子中， 技术，而不需要产生靶特异性sAB。为了测试和评估这些新的sAB模块，我们 将使用我们合作者网络的研究人员提供的一组高价值小膜蛋白。 这些系统已被排除在结构分析使用传统的方法，因此，将提供 一个很好的衡量伴侣辅助结构测定技术的性能。一个 重要副产品是这些结构将提供关于结构之间的连接的有价值的信息 以及以前无法实现的动态。 !

Abstract: 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 structure and 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 to be studied using traditional structural biology techniques. To overcome these barriers, we have developed and implemented a set of novel methodologies and reagents based on phage display generated synthetic antibodies (sABs). Customize phage display selection strategies enable generation of sABs endowed with special properties, for instance, conformation and regio-specificity. These reagents have been used to study the molecular properties of transient states of membrane proteins at unprecedented detail. While sABs have demonstrated efficacy as crystallization chaperones, their use in cryo-EM as powerful fiducial marks, adding 50 kDa to the particle and their ability to trap conformation states, is especially impactful in studies linking conformational transitions and function. This is particularly relevant for smaller membrane proteins (< 50 kDa), which include ion channels transporters and receptors. These constitute the largest class of biomedically relevant target systems, but are recalcitrant to crystallization and are far too small for cryo-EM analysis. Building on our current technology platform, we propose to design and deploy a set of higher-order sAB constructions that will serve to increase the size, rigidity and, in some cases, the symmetry of the target membrane protein. These sAB-based entities will be engineered to serve as prefabricated modules of assembly. They are targeted to specific epitopes that have been introduced into the membrane protein and thus, can be universally employed irrespective of the system they are applied to. The power of the approach is that these “universal” sABs can be added to the molecule of interest in a “plug and play” fashion allowing any investigator access to the powerful technology without requiring generating target specific sABs. To test and evaluate these novel sAB modules, we will use a set of high value small membrane proteins provided by investigators from our collaborator network. 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. An important byproduct is that these structures will provide valuable information about linkages between structure and dynamics that had been out of reach previously. !