Isotope labeling strategies for analysis of an ion channel cytoplasmic domain by NMR spectroscopy.
 

Isotope labeling strategies for analysis of an ion channel cytoplasmic domain by NMR spectroscopy.

Related Articles Isotope labeling strategies for analysis of an ion channel cytoplasmic domain by NMR spectroscopy.

Methods Mol Biol. 2013;998:289-300

Authors: Abarca-Heidemann K, Duchardt-Ferner E, Woehnert J, Rothberg BS

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.


PMID: 23529439 [PubMed - in process]



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