Quantification of Millisecond Protein-Folding Dynamics in Membrane-Mimetic Environments by Single-Molecule F枚rster Resonance Energy Transfer Spectroscopy

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• 作者：Andreas Hartmann ; Georg Krainer ; Sandro Keller ; Michael Schlierf
• 刊名：Analytical Chemistry
• 出版年：2015
• 出版时间：November 17, 2015
• 年：2015
• 卷：87
• 期：22
• 页码：11224-11232
• 全文大小：523K
• ISSN：1520-6882

文摘

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 F枚rster 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, semiquantitative, and quantitative analytical tools. Using this workflow, we analyzed urea-induced folding and unfolding experiments on the 伪-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.