title: Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels.
abstract: Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. Fundamental to these transitions is the intrinsic flexibility of the protein, which is critically modulated by membrane lipid-composition. To better understand the structural basis of channel function, it is necessary to study protein dynamics in a physiological membrane environment. Electron Paramagnetic Resonance (EPR) spectroscopy is an important tool to characterize conformational transitions between functional states. In comparison to NMR and X-ray crystallography, the information obtained from EPR is intrinsically of lower resolution. However, unlike in other techniques, in EPR there is no upper-limit to the molecular weight of the protein, the sample requirements are significantly lower, and more importantly the protein is not constrained by the crystal lattice forces. Therefore, EPR is uniquely suited for studying large protein complexes and proteins in reconstituted systems. In this article, we will discuss general protocols for site-directed spin labeling and membrane reconstitution using a prokaryotic proton-gated pentameric Ligand-Gated Ion Channel (pLGIC) from Gloeobacter violaceus (GLIC) as an example. A combination of steady-state Continuous Wave (CW) and Pulsed (Double Electron Electron Resonance-DEER) EPR approaches will be described that will enable a complete quantitative characterization of channel dynamics.
authors.affiliation: Department of Physiology and Biophysics, Case Western Reserve University.
authors.forename: Sandip
authors.initials: S
authors.lastname: Basak
authors.affiliation: Department of Physiology and Biophysics, Case Western Reserve University.
authors.forename: Soumili
authors.initials: S
authors.lastname: Chatterjee
authors.affiliation: Department of Physiology and Biophysics, Case Western Reserve University; School of Medicine, Case Western Reserve University; sxc584@case.edu.
authors.forename: Sudha
authors.initials: S
authors.lastname: Chakrapani
mesh_terms: D018360:Crystallography, X-Ray; D000458:Cyanobacteria; D004578:Electron Spin Resonance Spectroscopy; D015640:Ion Channel Gating; D058446:Ligand-Gated Ion Channels; D008958:Models, Molecular; D013113:Spin Labels
chemical_list: D058446:Ligand-Gated Ion Channels; D013113:Spin Labels