Isotope Labeling Strategies for Analysis of an Ion Channel Cytoplasmic Domain by NMR Spectroscopy

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.