Chaperone-Assisted Structure Determination of Membrane Proteins

分子伴侣辅助膜蛋白结构测定

• 批准号: 10321297
• 负责人: ANTHONY A KOSSIAKOFF
• 金额: $ 36.45万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10321297
• 关键词: Antibodies; Antibody Repertoire; Area; Binding; Binding Proteins; Biological; Biological Assay; Biological Process; Biology; Collaborations; Communities; Complex; Cryoelectron Microscopy; Crystallization; Custom; Development; Drug Industry; Drug Targeting; Elements; Engineering; Epitopes; Family member; Fruit; Funding; G Protein-Coupled Receptor Signaling; G protein coupled receptor kinase; G-Protein-Coupled Receptors; G-substrate; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Generations; Image; Immunoglobulin Fragments; Integral Membrane Protein; Ion Channel; Lead; Link; Measures; Membrane Proteins; Methodology; Methods; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; Molecular Machines; Nature; Parents; Pathogenesis; Performance; Phage Display; Phosphotransferases; Play; Productivity; Property; Protein Dynamics; Proteins; Reagent; Research; Research Personnel; Resistance; Resolution; Scientist; Services; Shapes; Signal Transduction; Site; Specificity; Structure; System; Techniques; Technology; Testing; Therapeutic; X-Ray Crystallography; base; cohort; conformational conversion; design; dimer; drug development; flexibility; insight; interest; novel; particle; programs; protein 50 kDa; protein complex; protein function; protein structure; public health relevance; receptor; structural biology; success; synthetic antibodies; tool; virtual

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. !

摘要： 膜蛋白是一种复杂的分子机器，其功能由一整套 程序化的构象转变。试图建立联系的基本分子机制 膜蛋白的结构和动力学对它们诱导的功能的影响受到了许多 看似不可逾越的技术障碍。这些障碍中的主要因素是构象转变 都是暂时的，不能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 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(Sabs)。自定义噬菌体展示选择策略支持生成赋予sabs 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 膜蛋白瞬变状态的分子性质具有前所未有的详细程度。而萨伯斯则有 展示了作为结晶伴侣的有效性，它们在冷冻-EM中作为强大的基准标记使用，增加了50% KDA对粒子及其捕获构象状态的能力，在研究连接 构象转变和功能。这与较小的膜蛋白(&lt；50 kDa)尤其相关， 包括离子通道、转运体和受体。它们构成了与生物医学相关的最大类别 目标系统，但不利于结晶，而且太小，无法进行低温电子显微镜分析。建立在我们的 目前的技术平台，我们建议设计和部署一套更高阶的SAB结构，将 用来增加目标膜蛋白的大小、刚性，在某些情况下，还可以增加其对称性。这些 基于SAB的实体将被设计为预制的组装模块。他们的目标是 被引入膜蛋白中的特定表位，因此可以被普遍使用 而不考虑它们所应用的系统。这种方法的威力在于，这些“通用”的sabs可以 以即插即用的方式添加到感兴趣的分子中，允许任何研究人员访问强大的 技术，而不需要生成目标特定的SAB。为了测试和评估这些新的SAB模块，我们 将使用由我们的合作者网络的研究人员提供的一套高价值的小膜蛋白。 这些系统一直拒绝使用传统方法进行结构分析，因此将提供 很好地衡量了伴侣辅助结构确定技术的性能。一个 重要的副产品是，这些结构将提供关于结构之间联系的有价值的信息 以及以前遥不可及的动力。 好了！