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

项目摘要 了解蛋白质折叠途径对理解蛋白质很重要 功能。蛋白质折叠可以在体外利用全长蛋白质在稀释液中进行研究 解决方案在为成功折叠而优化的条件下。然而，在建筑群中， 在细胞的环境中，蛋白质的共折叠和翻译后折叠 观察到的。共翻译折叠的矢量性质以及相互作用 在新生的链、修饰酶和分子伴侣之间 抑制不利的相互作用，如聚集和平滑能源格局， 从而使在细胞中折叠与在试管中折叠有很大不同。到目前为止， 关于细胞中新生的链所能获得的构象信息很少。 以及其相互作用的伙伴如何影响这些构象。这种知识上的差距是 主要是由于在细胞内观察折叠反应的实验困难。至 克服这一局限，我们的目标是开发一种新的方法来研究蛋白质折叠。这 方法，名为脉冲追逐细胞内蛋白质快速光化学氧化(PCIC- FPOP)将传统的脉冲追逐技术与基于质谱学的技术相结合。 单元格足迹方法。PCIC-FPOP将提供比凝胶更高的分辨率信息 电泳法，这是目前用于脉冲追逐分析的分析技术 数据，因为串联质谱仪可以提供关于氨基酸残基的信息- 水平。这种方法的发展需要重新设计足迹平台。 我们设计了一种新的细胞内足迹跟踪平台，其中包括一个舞台顶部 孵化器和纳米定位系统。我们将组装和优化新平台 目的：验证其对PCIC-FPOP的疗效(靶点1)。我们将使用Alpha 1 以抗胰蛋白酶(A1AT)为模型系统，检验该方法对短时荧光信号的探测能力。 活体折叠中间体(特定目标2)。我们还将研究A1AT的两个突变体S 和Z，以确定PCIC-FPOP是否可以检测到蛋白质错误折叠(特异性目标3)。 S突变体有轻微的折叠缺陷，而Z突变体有更严重的缺陷。 对这两种蛋白质的研究将决定该方法在检测中的灵敏度 蛋白质错误折叠。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。

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.