Chaperone-Assisted Structure Determination of Membrane Proteins

分子伴侣辅助膜蛋白结构测定

• 批准号: 9007806
• 负责人: ANTHONY A KOSSIAKOFF
• 金额: $ 34.29万
• 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 2019-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9007806
• 关键词: Advanced Development; Antibodies; Area; Biological; Biological Models; Biological Process; Biophysics; Chicago; Collaborations; Collection; Communities; Community Outreach; Complex; Crystallization; Crystallography; Detergents; Development; Drug Industry; Drug Targeting; Engineering; Enhancement Technology; Ensure; Environment; Generations; Guidelines; Health; Ion Channel; Knowledge; Lead; Libraries; Link; Lipids; Measures; Membrane; Membrane Proteins; Methodology; Methods; Molecular; Molecular Chaperones; Molecular Conformation; Molecular Machines; Mutagenesis; Performance; Phage Display; Process; Property; Protein Conformation; Protein Dynamics; Proteins; Protocols documentation; Reagent; Research; Research Infrastructure; Research Personnel; Resources; Roentgen Rays; Scientist; Services; Site; Sorting - Cell Movement; Stimulus; Structure; System; Techniques; Technology; Testing; Therapeutic; Universities; Work; antibody engineering; base; cohort; conformational conversion; empowered; interest; nanodisk; next generation; novel; particle; programs; protein complex; scaffold; structural biology; success; synthetic antibodies; tool; trait

 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.

 描述（应用程序提供）：膜蛋白是复杂的分子机器，其功能受编程会议过渡的组合。试图建立将膜蛋白动力学与影响的功能联系起来的基本分子机制的尝试受到了许多看似无法克服的技术障碍的挫败。这些障碍中的主要内容是，使用传统的结构生物学技术，构象过渡是太短暂的。为了克服这些障碍，提出了一项计划，以基于噬菌体显示生成的合成抗体（SABS）开发和实施一组新的方法和试剂，该抗体（SABS）提供了新的手段来研究膜蛋白瞬时状态的分子特性。这个概念是将这些SAB用作自定义的结晶伴侣或单个粒子冷冻EM应用的基准标记。为了推进这些发展，将结合两种强大的技术方法。第一项技术是一组新型的噬菌体展示排序策略，可以赋予SABS具有特殊属性，从而为调查人员提供了解决复杂的结构和生物学问题的工具，这些结构和生物学问题以前被认为太令人生畏，甚至无法考虑。第二种技术的进步是在充满脂质的纳米盘中进行这些选择，以更忠实地概括本地膜环境。该环境将更好地稳定由力引起的所需会议状态，因此，所选的SAB将捕获和稳定真正的动态中间体，然后可以通过X射线或冷冻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，将采用抗体工程来重塑SAB支架，以有效地多次多次多次从每个SAB粘合剂中得出的潜在伴侣数量。为了测试和评估方法，将使用三类膜离子通道作为模型系统。这些系统已经使用传统方法进行了结构分析，因此将很好地衡量伴侣辅助结构测定技术的性能。