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. !

抽象的： 膜蛋白是复杂的分子机器，其功能由一组 计划的过渡试图建立基本的分子机制 它们诱导的膜蛋白结构和动力学已被许多人挫败 看似叛乱的障碍物是这些障碍的校长 太过了，无法研究传统的结构技术。 已经根据生成的噬菌体显示开发并实施了一套新的方法和试剂 合成抗体（SABS）。 例如，使用特殊特性，这些试剂已用于研究。 膜蛋白的分子特性在未经前期的细节上 表现为结晶伴侣的功效，它们在冷冻EM中用作强大的基准标记，增加了50个 KDA到粒子和捕获混乱状态的三个能力，尤其是与之相关的研究。 构象过渡和功能与较小的膜蛋白（<50 kDa）特别相关， 包括离子通道转运蛋白和受体。 目标系统是结晶的重新晶体，并且对于在我们的基础上进行了太小 当前的技术平台，我们建议设计和部署一套高建筑 用于增加目标膜蛋白的大小，固定性和对称性。 基于SAB的实体将被发动机作为组装的预制模块。 已被成员雇用的特定表位 不管它是适用于该方法的力量。 以“插件”方式添加到感兴趣的分子中，使任何调查员都可以访问有力 技术不需要生成特定目标的SAB来测试和评估Thesenovel SAB模块 将使用我们协作者网络调查人员提供的一组高价值的小膜蛋白。 这些系统已经使用传统方法进行了结构分析，因此将提供 伴侣辅助结构确定技术的良好度量。 重要的副产品是结构提供有关结构之间联系的有价值信息的结构 以及以前无法触及的动态。 呢