title: Monitoring structural transitions in IDPs by site-directed spin labeling EPR spectroscopy.
abstract: Electron paramagnetic resonance (EPR) spectroscopy is a technique that specifically detects unpaired electrons. EPR sensitive reporter groups (spin labels or spin probes) can be introduced into biological systems via site-directed spin labeling (SDSL). This is usually accomplished by cysteine-substitution mutagenesis followed by covalent modification of the unique sulfhydryl group with a selective nitroxide reagent. SDSL EPR spectroscopy has been shown to be a sensitive and powerful method to study structural transitions within intrinsically disordered proteins (IDPs). In this chapter, we provide a detailed experimental protocol for this approach and present a few examples of EPR spectral shapes illustrative of various mobility regimes of the spin probe, reflecting different protein topologies.
authors: Architecture et Fonction des Macromolécules Biologiques, UMR 7257 CNRS and Aix-Marseille Université, Marseille, France.
authors: Johnny
authors: J
authors: Habchi
authors: Marlène
authors: M
authors: Martinho
authors: Antoine
authors: A
authors: Gruet
authors: Bruno
authors: B
authors: Guigliarelli
authors: Sonia
authors: S
authors: Longhi
authors: Valérie
authors: V
authors: Belle
mesh_terms: D000465:Algorithms; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli; D016297:Mutagenesis, Site-Directed; D009589:Nitrogen Oxides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D013194:Staining and Labeling; D013395:Sucrose / Q000737:chemistry; D014270:Trifluoroethanol / Q000737:chemistry
chemical_list: D009589:Nitrogen Oxides; D011993:Recombinant Fusion Proteins; D013395:Sucrose; D014270:Trifluoroethanol; C039900:nitroxyl; D003545:Cysteine