Development of a novel pulse-chase in-cell footprinting method for protein folding analysis

开发一种用于蛋白质折叠分析的新型脉冲追踪细胞内足迹方法

• 批准号: 9750170
• 负责人: Lisa M Jones
• 金额: $ 29.54万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9750170
• 关键词: Affect; Amino Acids; Benchmarking; Biological Models; Cell Culture Techniques; Cells; Complex; Coupled; Couples; Coupling; Custom; Data; Defect; Development; Environment; Enzymes; Exposure to; Gel; Goals; Hydrogen Peroxide; Hydroxyl Radical; In Vitro; Incidence; Incubators; Knowledge; Lasers; Length; Light; Mass Spectrum Analysis; Methods; Molecular; Molecular Chaperones; Molecular Conformation; Movement; Mutation; Nature; Pathway interactions; Physiologic pulse; Protein-Folding Disease; Proteins; Reaction; Resolution; Sampling; Solvents; Structural Protein; System; Techniques; Technology; Testing; Time; Transfection; Tube; activator 1 protein; alpha 1-Antitrypsin; base; design; detector; experimental study; gel electrophoresis; method development; mutant; novel; oxidation; photolysis; protein folding; protein function; protein misfolding; protein structure; tandem mass spectrometry; tool; vector

Project Summary An understanding of protein folding pathways is important in understanding protein function. Protein folding can be studied in vitro using full-length proteins in dilute solutions under conditions optimized for successful folding. However, in the complex environment of the cell, co- and post-translational folding of proteins has been observed. The vectorial nature of co-translational folding as well as interactions between the nascent chain, modifying enzymes, and molecular chaperones presumably inhibit unfavorable interactions such as aggregation and smooth the energy landscape, thereby making folding in the cell quite different from folding in the test-tube. To date, there is a dearth of information about conformations available to a nascent chain in cells and how its interacting partners affect these conformations. This gap in knowledge is mainly due to the experimental difficulty of observing the folding reaction in cells. To overcome this limitation, we aim to develop a new method for study protein folding. This method, entitled pulse-chase in-cell fast photochemical oxidation of proteins (pcIC- FPOP) couples traditional pulse-chase technology with a mass spectrometry-based in- cell footprinting method. pcIC-FPOP will provide higher resolution information than gel electrophoresis, which is the current analytical technique for analysis of pulse-chase data, as tandem mass spectrometry can provide information on the amino acid residue- level. The development of this method requires a redesign of the footprinting platform. We have designed a new platform for in-cell footprinting that includes a stage-top incubator and nanopositioning system. We will assemble and optimize the new platform to demonstrate its efficacy for pcIC-FPOP (specific aim 1). We will use alpha 1 antitrypsin (A1AT) as a model system to test the ability of the method to probe short- lived folding intermediates (specific aim 2). We will also study two mutants of A1AT, S and Z to determine whether pcIC-FPOP can detect protein misfolding (specific aim 3). The S mutant has a mild folding defect while the Z mutant has a more severe defect. The study of both proteins will determine the sensitivity of the method in detecting protein misfolding. The developed method would provide a new, higher resolution tool for studying protein folding in the native cellular environment.

项目摘要 蛋白质折叠途径的理解对于理解蛋白质是重要的 功能蛋白质折叠可以在体外研究，使用全长蛋白质在稀溶液中。 在为成功折叠而优化的条件下获得解决方案。然而，在复杂的 在细胞环境中，蛋白质的共翻译和翻译后折叠已经被 观察共翻译折叠的矢量性质以及相互作用 在新生链、修饰酶和分子伴侣之间， 抑制不利的相互作用，如聚集和平滑的能源景观， 从而使细胞中的折叠与试管中的折叠完全不同。到目前为止， 关于细胞中新生链可用的构象信息缺乏 以及它的相互作用伙伴如何影响这些构象。这种知识差距是 主要是由于观察细胞中折叠反应的实验困难。到 本文旨在克服这一局限性，发展一种研究蛋白质折叠的新方法。这 方法，题为脉冲追逐细胞内快速光化学氧化的蛋白质（pcIC- FPOP）将传统的脉冲追踪技术与基于质谱的信息技术相结合， 细胞足迹法pcIC-FPOP将提供比凝胶更高的分辨率信息 电泳，这是目前用于分析脉冲追踪的分析技术 数据，因为串联质谱法可以提供关于氨基酸残基的信息- 水平开发这种方法需要重新设计足迹平台。 我们已经设计了一个新的平台，在细胞足迹，其中包括一个阶段的顶部 培养箱和纳米定位系统。我们将组装和优化新平台 证明其对pcIC-FPOP的有效性（具体目标1）。我们将使用alpha 1 抗胰蛋白酶（A1 AT）作为模型系统，以测试该方法探测短- 活的折叠中间体（具体目标2）。我们还将研究A1 AT的两个突变体，S 和Z，以确定pcIC-FPOP是否可以检测蛋白质错误折叠（特异性目的3）。 S突变体具有轻度折叠缺陷，而Z突变体具有更严重的缺陷。 对这两种蛋白质的研究将确定该方法在检测 蛋白质错误折叠所开发的方法将提供一种新的、更高分辨率的工具 用于研究天然细胞环境中的蛋白质折叠。