title: Preparation of uniformly isotope labeled KcsA for solid state NMR: expression, purification, reconstitution into liposomes and functional assay.
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.
authors.affiliation: Department of Chemistry, Columbia University, NY 10027, United States. Electronic address: mpb2124@columbia.edu.
authors.forename: Manasi P
authors.initials: MP
authors.lastname: Bhate
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Wylie
authors.forename: Ameer
authors.initials: A
authors.lastname: Thompson
authors.forename: Lin
authors.initials: L
authors.lastname: Tian
authors.forename: Crina
authors.initials: C
authors.lastname: Nimigean
authors.forename: Ann E
authors.initials: AE
authors.lastname: McDermott
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007553:Isotope Labeling; D008081:Liposomes / Q000737:chemistry* / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D015221:Potassium Channels / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D008081:Liposomes; D015221:Potassium Channels; D011994:Recombinant Proteins; C107790:prokaryotic potassium channel
keywords: Isotopic labeling; Liposomes; Membrane proteins; Reconstitution; Solid-state NMR
references.citation: J Am Chem Soc. 2008 Jun 11;130(23):7427-35
references.pmid: 18479093
references.citation: EMBO J. 1995 Nov 1;14(21):5170-8
references.pmid: 7489706
references.citation: Biophys J. 2008 Mar 1;94(5):1689-98
references.pmid: 18024500
references.citation: J Gen Physiol. 2002 Sep;120(3):323-35
references.pmid: 12198089
references.citation: J Biol Chem. 2007 May 18;282(20):15179-86
references.pmid: 17360718
references.citation: Protein Sci. 2006 Apr;15(4):684-98
references.pmid: 16522799
references.citation: Science. 1998 Apr 3;280(5360):69-77
references.pmid: 9525859
references.citation: Nat Struct Mol Biol. 2007 Nov;14(11):1089-95
references.pmid: 17922011
references.citation: Annu Rev Physiol. 2004;66:131-59
references.pmid: 14977399
references.citation: FEBS Lett. 2002 Aug 14;525(1-3):33-8
references.pmid: 12163157
references.citation: Proc Natl Acad Sci U S A. 2012 Sep 18;109(38):15265-70
references.pmid: 22942391
references.citation: J Gen Physiol. 1998 Jun;111(6):741-9
references.pmid: 9607934
references.citation: Proc Natl Acad Sci U S A. 2008 May 13;105(19):6900-5
references.pmid: 18443286
references.citation: Biochemistry. 1995 Jul 4;34(26):8309-19
references.pmid: 7599123
references.citation: J Magn Reson. 2000 Jan;142(1):97-101
references.pmid: 10617439
references.citation: J Gen Physiol. 2001 Sep;118(3):303-14
references.pmid: 11524460
references.citation: Chembiochem. 2007 Mar 5;8(4):434-42
references.pmid: 17285659
references.citation: Nature. 2010 Jul 8;466(7303):272-5
references.pmid: 20613845
references.citation: Nature. 2001 Nov 1;414(6859):43-8
references.pmid: 11689936
references.citation: Biochemistry. 1997 Aug 19;36(33):10335-42
references.pmid: 9254633
references.citation: J Biomol NMR. 2001 May;20(1):71-5
references.pmid: 11430757
references.citation: Anal Biochem. 1989 Nov 1;182(2):319-26
references.pmid: 2610349
references.citation: J Mol Biol. 2010 Aug 13;401(2):155-66
references.pmid: 20600123