title: Quantification of Millisecond Protein-Folding Dynamics in Membrane-Mimetic Environments by Single-Molecule Förster Resonance Energy Transfer Spectroscopy.
abstract: 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 Förster 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 α-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.
authors.affiliation: B CUBE-Center for Molecular Bioengineering, Technische Universität Dresden , Arnoldstr. 18, 01307 Dresden, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Hartmann
authors.affiliation: B CUBE-Center for Molecular Bioengineering, Technische Universität Dresden , Arnoldstr. 18, 01307 Dresden, Germany.|Molecular Biophysics, University of Kaiserslautern , Erwin-Schrödinger-Str. 13, 67663 Kaiserslautern, Germany.
authors.forename: Georg
authors.initials: G
authors.lastname: Krainer
authors.affiliation: Molecular Biophysics, University of Kaiserslautern , Erwin-Schrödinger-Str. 13, 67663 Kaiserslautern, Germany.
authors.forename: Sandro
authors.initials: S
authors.lastname: Keller
authors.affiliation: B CUBE-Center for Molecular Bioengineering, Technische Universität Dresden , Arnoldstr. 18, 01307 Dresden, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Schlierf
mesh_terms: D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry*; D018613:Microscopy, Confocal; D008958:Models, Molecular; D017510:Protein Folding; D013816:Thermodynamics
chemical_list: D008565:Membrane Proteins