title: Characterization of the disordered-to-α-helical transition of IA₃ by SDSL-EPR spectroscopy.
abstract: Electron paramagnetic resonance (EPR) spectroscopy coupled with site-directed spin labeling (SDSL) is a valuable tool for characterizing the mobility and conformational changes of proteins but has seldom been applied to intrinsically disordered proteins (IDPs). Here, IA₃ is used as a model system demonstrating SDSL-EPR characterization of conformational changes in small IDP systems. IA₃ has 68 amino acids, is unstructured in solution, and becomes α-helical upon addition of the secondary structural stabilizer 2,2,2-trifluoroethanol (TFE). Two single cysteine substitutions, one in the N-terminus (S14C) and one in the C-terminus (N58C), were generated and labeled with three different nitroxide spin labels. The resultant EPR line shapes of each of the labels were compared and each reported changes in mobility upon addition of TFE. Specifically, the spectral line shape parameters h((+1))/h(₀), the local tumbling volume (V(L)), and the percent change of the h(₋₁) intensity were utilized to quantitatively monitor TFE-induced conformational changes. The values of h((+1)/)h(₀) as a function of TFE titration varied in a sigmoidal manner and were fit to a two-state Boltzmann model that provided values for the midpoint of the transition, thus, reporting on the global conformational change of IA₃. The other parameters provide site-specific information and show that S14C-SL undergoes a conformational change resulting in more restricted motion than N58C-SL, which is consistent with previously published results obtained by studies using NMR and circular dichroism spectroscopy indicating a higher degree of α-helical propensity of the N-terminal segment of IA₃. Overall, the results provide a framework for data analyzes that can be used to study induced unstructured-to-helical conformations in IDPs by SDSL.
authors.affiliation: Department of Chemistry, University of Florida, Gainesville, Florida 32611, USA.
authors.forename: Natasha L
authors.initials: NL
authors.lastname: Pirman
authors.forename: Eugene
authors.initials: E
authors.lastname: Milshteyn
authors.forename: Luis
authors.initials: L
authors.lastname: Galiano
authors.forename: Justin C
authors.initials: JC
authors.lastname: Hewlett
authors.forename: Gail E
authors.initials: GE
authors.lastname: Fanucci
mesh_terms: D004578:Electron Spin Resonance Spectroscopy; D009589:Nitrogen Oxides / Q000737:chemistry; D017433:Protein Structure, Secondary; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D013113:Spin Labels
chemical_list: D009589:Nitrogen Oxides; C018134:PAI3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D013113:Spin Labels; C039900:nitroxyl