期刊
ANALYTICAL CHEMISTRY
卷 87, 期 22, 页码 11224-11232出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.5b03207
关键词
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资金
- German Federal Ministry of Education and Research BMBF [03Z2EN11, 03Z2ES1]
- Deutsche Forschungsgemeinschaft (DFG) [KE 1478/1-2]
- Stipendienstiftung Rheinland-Pfalz
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.
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