Quantification of Millisecond Protein-Folding Dynamics in Membrane-Mimetic Environments by Single-Molecule Forster Resonance Energy Transfer Spectroscopy
Quantification of Millisecond Protein-Folding Dynamics in Membrane-Mimetic Environments by Single-Molecule Forster Resonance Energy Transfer Spectroscopy
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DOI：
10.1021/acs.analchem.5b03207
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发表时间：
2015-11-17
影响因子：
7.4
通讯作者：
Schlierf, Michael
中科院分区：
文献类型：
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作者：
Hartmann, Andreas;Krainer, Georg;Schlierf, Michael
越来越多的膜蛋白在不同的膜模拟系统已成为可访问的可逆展开实验监测完善的合奏技术。然而，只有很少的信息是关于膜蛋白折叠过程中的动力学过程，主要是因为实验的挑战和缺乏适合于观察高度动态的膜蛋白的方法。在这里，我们提出了单分子福斯特共振能量转移（smFRET）共聚焦光谱作为一个强大的工具，在动力学研究膜蛋白折叠在膜模拟环境。我们已经开发了一个严格的工作流程，展示了如何使用一套定性，半定量和定量分析工具来识别和量化这些动态过程。使用这个工作流程，我们分析了尿素诱导的折叠和展开实验的α-螺旋膜蛋白Mistic的存在下的两性离子洗涤剂n-十二烷基磷酸胆碱（DPC）。我们确定了两个国家的相互转换动力学上的毫秒时间尺度的蛋白质折叠成和出洗涤剂胶束。我们的研究结果表明，smFRET是一个很有前途的工具，用于探测化学物理的膜蛋白质的结构和动力学的复杂和各向异性的环境中的亲水/疏水界面，提供洞察蛋白质相互转换的动力学，而不需要和同步的挑战。
An increasing number of membrane proteins in different membrane-mimetic systems have become accessible to reversible unfolding experiments monitored by well-established ensemble techniques. However, only little information is available about kinetic processes during membrane-protein folding, mainly because of experimental challenges and a lack of methods suitable for observing highly dynamic membrane proteins. Here, we present single-molecule Forster resonance energy transfer (smFRET) confocal spectroscopy as a powerful tool in kinetic studies of membrane-protein folding in membrane-mimetic environments. We have developed a rigorous workflow demonstrating how to identify and quantify such dynamic processes using a set of qualitative, semi-quantitative, and quantitative analytical tools. Using this workflow, we analyzed urea-induced folding and unfolding experiments on the a-helical membrane protein Mistic in the presence of the zwitterionic detergent n-dodecylphosphocholine (DPC). We identified two-state interconversion dynamics on the millisecond time scale of a protein folding into and out of detergent micelles. Our results demonstrate that smFRET is a promising tool for probing the chemical physics of membrane-protein structure and dynamics in the complex and anisotropic environment of a hydrophilic/hydrophobic interface, providing insights into protein interconversion dynamics without the need and challenges of synchronization.