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Preparation of uniformly isotope labeled KcsA for solid state NMR: expression, purification, reconstitution into liposomes and functional assay.

TitlePreparation of uniformly isotope labeled KcsA for solid state NMR: expression, purification, reconstitution into liposomes and functional assay.
Publication TypeJournal Article
Year of Publication2013
AuthorsBhate MP, Wylie BJ, Thompson A, Tian L, Nimigean C, McDermott AE
JournalProtein Expr Purif
Volume91
Issue2
Pagination119-24
Date Published2013 Oct
ISSN1096-0279
Abstract

We report the expression, purification, liposome reconstitution and functional validation of uniformly (13)C and (15)N isotope labeled KcsA, a bacterial potassium channel that has high homology with mammalian channels, for solid-state NMR studies. The expression and purification is optimized for an average yield of ∼35-40mg/L of M9 media in a time-efficient way. The protein purity is confirmed by gel electrophoresis and the protein concentration is quantified by UV-vis absorption spectroscopy. Protocols to efficiently reconstitute KcsA into liposomes are also presented. The presence of liposomes is confirmed by cryo-electron microscopy images and the effect of magic angle spinning on liposome packing is shown. High-resolution solid-state NMR spectra of uniformly isotope labeled KcsA in these liposomes reveal that our protocol yields to a very homogenous KcsA sample with high signal to noise and several well-resolved residues in NMR spectra. Electrophysiology of our samples before and after solid-state NMR show that channel function and selectivity remain intact after the solid-state NMR.

DOI10.1016/j.pep.2013.07.013
Alternate JournalProtein Expr. Purif.
PubMed ID23916531
PubMed Central IDPMC3805054
Grant ListF32 087908 / / PHS HHS / United States
P41GM66354 / GM / NIGMS NIH HHS / United States
R01 GM088724 / GM / NIGMS NIH HHS / United States
R01 GM88724 / GM / NIGMS NIH HHS / United States
R01GM088352 / GM / NIGMS NIH HHS / United States