title: Identification and removal of nitroxide spin label contaminant: impact on PRE studies of α-helical membrane proteins in detergent.
abstract: NMR paramagnetic relaxation enhancement (PRE) provides long-range distance constraints (~15-25 Å) that can be critical to determining overall protein topology, especially where long-range NOE information is unavailable such as in the case of larger proteins that require deuteration. However, several challenges currently limit the use of NMR PRE for α-helical membrane proteins. One challenge is the nonspecific association of the nitroxide spin label to the protein-detergent complex that can result in spurious PRE derived distance restraints. The effect of the nitroxide spin label contaminant is evaluated and quantified and a robust method for the removal of the contaminant is provided to advance the application of PRE restraints to membrane protein NMR structure determination.
authors.affiliation: Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, USA.
authors.forename: Brett M
authors.initials: BM
authors.lastname: Kroncke
authors.forename: Linda
authors.initials: L
authors.lastname: Columbus
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D002462:Cell Membrane / Q000737:chemistry; D003001:Cloning, Molecular; D003497:Cyclic N-Oxides / Q000737:chemistry; D003902:Detergents / Q000737:chemistry*; D005798:Genes, Bacterial; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry*; D008698:Mesylates / Q000737:chemistry; D046912:Multiprotein Complexes / Q000737:chemistry; D017433:Protein Structure, Secondary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013113:Spin Labels; D020124:Thermotoga maritima / Q000737:chemistry / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D003497:Cyclic N-Oxides; D003902:Detergents; D008565:Membrane Proteins; D008698:Mesylates; D046912:Multiprotein Complexes; D013113:Spin Labels; C034248:(1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)methanethiosulfonate