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

Manasi P. Bhate, Benjamin J. Wylie, Ameer Thompson, Lin Tian, Crina Nimigean, Ann E. McDermott

Research output: Contribution to journalArticle

15 Scopus citations

Abstract

We report the expression, purification, liposome reconstitution and functional validation of uniformly 13C and 15N 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 ∼35-40 mg/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.

Original languageEnglish
Pages (from-to)119-124
Number of pages6
JournalProtein Expression and Purification
Volume91
Issue number2
DOIs
StatePublished - 2013

Keywords

  • Isotopic labeling
  • Liposomes
  • Membrane proteins
  • NMR
  • Reconstitution
  • Solid-state

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