title: A rigid disulfide-linked nitroxide side chain simplifies the quantitative analysis of PRE data.
abstract: The measurement of (1)H transverse paramagnetic relaxation enhancement (PRE) has been used in biomolecular systems to determine long-range distance restraints and to visualize sparsely-populated transient states. The intrinsic flexibility of most nitroxide and metal-chelating paramagnetic spin-labels, however, complicates the quantitative interpretation of PREs due to delocalization of the paramagnetic center. Here, we present a novel, disulfide-linked nitroxide spin label, R1p, as an alternative to these flexible labels for PRE studies. When introduced at solvent-exposed α-helical positions in two model proteins, calmodulin (CaM) and T4 lysozyme (T4L), EPR measurements show that the R1p side chain exhibits dramatically reduced internal motion compared to the commonly used R1 spin label (generated by reacting cysteine with the spin labeling compound often referred to as MTSL). Further, only a single nitroxide position is necessary to account for the PREs arising from CaM S17R1p, while an ensemble comprising multiple conformations is necessary for those observed for CaM S17R1. Together, these observations suggest that the nitroxide adopts a single, fixed position when R1p is placed at solvent-exposed α-helical positions, greatly simplifying the interpretation of PRE data by removing the need to account for the intrinsic flexibility of the spin label.
authors.affiliation: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MA 20892-0520, USA.
authors.forename: Nicolas L
authors.initials: NL
authors.lastname: Fawzi
authors.forename: Mark R
authors.initials: MR
authors.lastname: Fleissner
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Anthis
authors.forename: Tamás
authors.initials: T
authors.lastname: Kálai
authors.forename: Kálmán
authors.initials: K
authors.lastname: Hideg
authors.forename: Wayne L
authors.initials: WL
authors.lastname: Hubbell
authors.forename: G Marius
authors.initials: GM
authors.lastname: Clore
mesh_terms: D017122:Bacteriophage T4 / Q000201:enzymology; D002147:Calmodulin / Q000737:chemistry*; D003497:Cyclic N-Oxides / Q000138:chemical synthesis / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D006801:Humans; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry*; D011487:Protein Conformation; D013113:Spin Labels / Q000138:chemical synthesis
chemical_list: D002147:Calmodulin; D003497:Cyclic N-Oxides; D004220:Disulfides; D013113:Spin Labels; D009113:Muramidase