title: Site-directed electrostatic measurements with a thiol-specific ph-sensitive nitroxide: differentiating local pK and polarity effects by high-field EPR.
abstract: This communication describes the use of a methanethiosulfonate derivative of an imidazolidine nitroxide, methanethiosulfonic acid S-(1-oxyl-2,2,3,5,5-pentamethyl-imidazolidin-4-ylmethyl) ester, IMTSL, for site-directed pKa determination of peptides by electron paramagnetic resonance. This spin label is covalently attached to the thiol group of unique cysteines incorporated into peptide structures. The tertiary amine nitrogen N3 of the label readily participates in proton exchange reactions, which are monitored through changes in EPR spectra of nitroxide moiety. Using EPR at 95 GHz (W-band) isotropic magnetic parameters of this nitroxide, both Aiso and giso, were calibrated in solvents of different polarity and pH. Two different linear correlations between Aiso and giso for acidic and basic forms of IMTSL were observed, making it possible to differentiate effects of local polarity from N3 protonation on nitroxide EPR spectra. Titration of a synthetic P11 peptide fragment of the laminin B1 chain illustrates the utility of this method.
authors.affiliation: Department of Chemistry, North Carolina State University, 2620 Yarbrough Drive, Raleigh, North Carolina 27695-8204, USA. alex_smirnov@ncsu.edu
authors.forename: Alex I
authors.initials: AI
authors.lastname: Smirnov
authors.forename: Andres
authors.initials: A
authors.lastname: Ruuge
authors.forename: Vladimir A
authors.initials: VA
authors.lastname: Reznikov
authors.forename: Maxim A
authors.initials: MA
authors.lastname: Voinov
authors.forename: Igor A
authors.initials: IA
authors.lastname: Grigor'ev
mesh_terms: D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000737:chemistry; D007700:Kinetics; D008698:Mesylates / Q000737:chemistry; D009589:Nitrogen Oxides / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D017510:Protein Folding; D013113:Spin Labels; D055672:Static Electricity; D013438:Sulfhydryl Compounds / Q000737:chemistry*
chemical_list: D007093:Imidazoles; D008698:Mesylates; D009589:Nitrogen Oxides; D010455:Peptides; D013113:Spin Labels; D013438:Sulfhydryl Compounds; C074502:methanethiosulfonate