title: Isotope labeling strategies for analysis of an ion channel cytoplasmic domain by NMR spectroscopy.
abstract: As large, multimeric, integral membrane proteins, ion channels pose technical challenges to analysis by NMR spectroscopy. Here we present a strategy to overcome some of these technical hurdles, using a representative ion channel modulatory domain, the regulator of K(+) conductance (RCK) domain from a K(+) channel cloned from Thermoplasma volcanium. By introducing a mutation to limit the stoichiometry of the octameric RCK domain "gating ring" complex to its dimeric building block, NMR spectral resolution can be greatly improved. Here we present protocols for efficient production of highly deuterated, uniformly (15)N-labeled protein, as well as protein containing (15)N-labeling to specific amino acid types. These labeling strategies can be applied to improve spectral resolution and facilitate sequential resonance assignments.
authors.affiliation: Department of Biochemistry, Temple University School of Medicine, Philadelphia, PA, USA.
authors.forename: Karin
authors.initials: K
authors.lastname: Abarca-Heidemann
authors.forename: Elke
authors.initials: E
authors.lastname: Duchardt-Ferner
authors.forename: Jens
authors.initials: J
authors.lastname: Woehnert
authors.forename: Brad S
authors.initials: BS
authors.lastname: Rothberg
mesh_terms: D003593:Cytoplasm / Q000378:metabolism*; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics; D007473:Ion Channels / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007553:Isotope Labeling / Q000379:methods*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D017434:Protein Structure, Tertiary; D014170:Transformation, Genetic
chemical_list: D007473:Ion Channels