Chaperone-Assisted Structure Determination of Membrane Proteins

分子伴侣辅助膜蛋白结构测定

• 批准号: 9887438
• 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/9887438
• 关键词: Antibodies; Antibody Repertoire; Area; Binding; Binding Proteins; Biological; Biological Assay; Biological Process; 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. !

抽象的： 膜蛋白是复杂的分子机器，其功能由一组控制 程序化构象转变。尝试建立联系的基本分子机制 膜蛋白的结构和它们诱导的功能动力学已受到许多因素的阻碍 看似难以逾越的技术障碍。这些障碍中最主要的是构象转变 它们太短暂，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，我们 开发并实施了一套基于噬菌体展示的新颖方法和试剂 合成抗体（sAB）。定制噬菌体展示选择策略能够生成赋予的 sAB 具有特殊的性质，例如构象和区域特异性。这些试剂已用于研究 以前所未有的细节描述膜蛋白瞬态的分子特性。虽然 sAB 有 证明了作为结晶伴侣的功效，它们在冷冻电镜中用作强大的基准标记，增加了 50 粒子的 kDa 及其捕获构象状态的能力，在链接的研究中特别有影响力 构象转变和功能。这对于较小的膜蛋白 (< 50 kDa) 尤其相关， 其中包括离子通道转运蛋白和受体。这些构成了生物医学相关的最大类别 目标系统，但难以结晶，并且对于冷冻电镜分析来说太小。建立在我们的 在当前的技术平台上，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，在某些情况下还增加其对称性。这些 基于 sAB 的实体将被设计为用作装配的预制模块。他们的目标是 已引入膜蛋白中的特定表位，因此可以普遍使用 无论它们应用于什么系统。该方法的强大之处在于这些“通用”sAB 可以 以“即插即用”的方式添加到感兴趣的分子中，允许任何调查人员访问强大的 技术，无需生成目标特定的 SAB。为了测试和评估这些新颖的 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。 这些系统一直难以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测定技术的性能。一个 重要的副产品是这些结构将提供有关结构之间联系的有价值的信息 以及以前无法达到的动态。 ！