title: Combination of ¹⁵N reverse labeling and afterglow spectroscopy for assigning membrane protein spectra by magic-angle-spinning solid-state NMR: application to the multidrug resistance protein EmrE.
abstract: Magic-angle-spinning (MAS) solid-state NMR spectroscopy has emerged as a viable method to characterize membrane protein structure and dynamics. Nevertheless, the spectral resolution for uniformly labeled samples is often compromised by redundancy of the primary sequence and the presence of helical secondary structure that results in substantial resonance overlap. The ability to simplify the spectrum in order to obtain unambiguous site-specific assignments is a major bottleneck for structure determination. To address this problem, we used a combination of (15)N reverse labeling, afterglow spectroscopic techniques, and frequency-selective dephasing experiments that dramatically improved the ability to resolve peaks in crowded spectra. This was demonstrated using the polytopic membrane protein EmrE, an efflux pump involved in multidrug resistance. Residues preceding the (15)N reverse labeled amino acid were imaged using a 3D NCOCX afterglow experiment and those following were recorded using a frequency-selective dephasing experiment. Our approach reduced the spectral congestion and provided a sensitive way to obtain chemical shift assignments for a membrane protein where no high-resolution structure is available. This MAS methodology is widely applicable to the study of other polytopic membrane proteins in functional lipid bilayer environments.
authors.affiliation: Department of Chemistry, New York University, 100 Washington Square East, New York, NY 10003, USA.
authors.forename: James R
authors.initials: JR
authors.lastname: Banigan
authors.forename: Anindita
authors.initials: A
authors.lastname: Gayen
authors.forename: Nathaniel J
authors.initials: NJ
authors.lastname: Traaseth
mesh_terms: D017920:Antiporters / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008954:Models, Biological
chemical_list: D017920:Antiporters; D029968:Escherichia coli Proteins; C075436:EmrE protein, E coli
references.citation: Biochim Biophys Acta. 2012 Feb;1818(2):146-53
references.pmid: 21839724
references.citation: J Am Chem Soc. 2004 Jun 2;126(21):6720-7
references.pmid: 15161300
references.citation: Structure. 2011 Dec 7;19(12):1732-8
references.pmid: 22153495
references.citation: J Biomol NMR. 2007 Oct;39(2):107-31
references.pmid: 17687624
references.citation: J Am Chem Soc. 2006 Mar 15;128(10):3154-5
references.pmid: 16522090
references.citation: J Phys Chem B. 2012 Jun 21;116(24):7138-44
references.pmid: 22582831
references.citation: J Magn Reson. 2003 Jun;162(2):479-86
references.pmid: 12810033
references.citation: J Mol Biol. 2009 Mar 6;386(4):1078-93
references.pmid: 19244620
references.citation: J Mol Biol. 2006 Nov 17;364(1):54-67
references.pmid: 17005200
references.citation: J Biomol NMR. 2008 Jul;41(3):121-5
references.pmid: 18516685
references.citation: J Magn Reson. 2006 Dec;183(2):324-8
references.pmid: 16990042
references.citation: Proc Natl Acad Sci U S A. 2012 Sep 18;109(38):15265-70
references.pmid: 22942391
references.citation: Proc Natl Acad Sci U S A. 2011 May 31;108(22):9101-6
references.pmid: 21576492
references.citation: Prog Nucl Magn Reson Spectrosc. 2009 Nov 1;55(4):335-360
references.pmid: 20161395
references.citation: Angew Chem Int Ed Engl. 2012 Mar 12;51(11):2731-5
references.pmid: 22311700
references.citation: J Biol Chem. 2008 Feb 8;283(6):3281-3288
references.pmid: 18042544
references.citation: J Biomol NMR. 1999 May;14(1):71-4
references.pmid: 10382307
references.citation: J Mol Biol. 2010 Mar 19;397(1):301-15
references.pmid: 20080102
references.citation: J Am Chem Soc. 2010 Apr 28;132(16):5558-9
references.pmid: 20356036
references.citation: J Am Chem Soc. 2004 Sep 22;126(37):11422-3
references.pmid: 15366872
references.citation: Protein Sci. 2012 Jan;21(1):84-96
references.pmid: 22095702
references.citation: Annu Rev Phys Chem. 2011;62:279-99
references.pmid: 21219138
references.citation: J Biomol NMR. 2011 Jan;49(1):39-51
references.pmid: 21153044
references.citation: Chembiochem. 2010 Jul 26;11(11):1543-51
references.pmid: 20572250
references.citation: Biochemistry. 2006 Nov 21;45(46):13827-34
references.pmid: 17105201
references.citation: J Biol Chem. 2012 Jan 27;287(5):3479-84
references.pmid: 22147705
references.citation: Acta Crystallogr D Biol Crystallogr. 2009 Feb;65(Pt 2):186-92
references.pmid: 19171974
references.citation: Proc Natl Acad Sci U S A. 2004 Jan 20;101(3):711-6
references.pmid: 14715898
references.citation: Biochim Biophys Acta. 2009 May;1794(5):748-62
references.pmid: 19167526
references.citation: Nature. 2011 Jul 19;477(7366):549-55
references.pmid: 21772288
references.citation: Proc Natl Acad Sci U S A. 2007 Nov 27;104(48):18999-9004
references.pmid: 18024586
references.citation: J Am Chem Soc. 2011 Dec 7;133(48):19266-9
references.pmid: 22039931
references.citation: Nature. 2011 Dec 18;481(7379):45-50
references.pmid: 22178925
references.citation: Proc Natl Acad Sci U S A. 2009 Jun 23;106(25):10165-70
references.pmid: 19509339
references.citation: Mol Cell. 2008 Sep 26;31(6):896-908
references.pmid: 18922471
references.citation: J Biol Chem. 2011 Aug 19;286(33):28988-28995
references.pmid: 21715337
references.citation: Chembiochem. 2012 Mar 19;13(5):732-9
references.pmid: 22408059
references.citation: Science. 2010 Oct 22;330(6003):509-12
references.pmid: 20966252
references.citation: Nature. 2002 Nov 7;420(6911):98-102
references.pmid: 12422222
references.citation: Proc Natl Acad Sci U S A. 2007 Oct 9;104(41):16140-5
references.pmid: 17911261
references.citation: Annu Rev Phys Chem. 2012;63:1-24
references.pmid: 22136620
references.citation: J Am Chem Soc. 2008 Jun 25;130(25):7818-9
references.pmid: 18512910
references.citation: J Am Chem Soc. 2006 Jun 14;128(23):7402-3
references.pmid: 16756269
references.citation: Proc Natl Acad Sci U S A. 2012 Mar 27;109(13):4863-8
references.pmid: 22331896
references.citation: Angew Chem Int Ed Engl. 2007;46(3):459-62
references.pmid: 17001715
references.citation: Biochim Biophys Acta. 2007 Dec;1768(12):3036-43
references.pmid: 17976529
references.citation: J Am Chem Soc. 2008 Apr 30;130(17):5798-807
references.pmid: 18393505
references.citation: Nature. 2009 May 21;459(7245):344-6
references.pmid: 19458709
references.citation: Science. 2008 Aug 29;321(5893):1206-10
references.pmid: 18755977
references.citation: J Biomol NMR. 2008 Apr;40(4):239-50
references.pmid: 18320329
references.citation: Protein Sci. 2012 Nov;21(11):1620-33
references.pmid: 23001990
references.citation: Chemphyschem. 2011 Aug 1;12(11):2092-6
references.pmid: 21656893
references.citation: J Biomol NMR. 2012 Dec;54(4):343-53
references.pmid: 23053913
references.citation: Nature. 2010 Feb 4;463(7281):689-92
references.pmid: 20130653
references.citation: J Am Chem Soc. 2010 Jun 23;132(24):8255-7
references.pmid: 20509649
references.citation: Adv Exp Med Biol. 2012;992:35-62
references.pmid: 23076578
references.citation: J Am Chem Soc. 2006 Sep 20;128(37):12256-67
references.pmid: 16967977
references.citation: J Magn Reson. 2011 Jul;211(1):18-24
references.pmid: 21482162
references.citation: J Biol Chem. 2010 Aug 20;285(34):26710-8
references.pmid: 20551331
references.citation: J Biol Chem. 2010 Oct 8;285(41):31723-30
references.pmid: 20667827
references.citation: Protein Sci. 1998 Apr;7(4):1029-38
references.pmid: 9568909
references.citation: Nature. 2012 Jan 09;481(7382):469-74
references.pmid: 22230955
references.citation: Proc Natl Acad Sci U S A. 2005 Nov 1;102(44):15871-6
references.pmid: 16247008
references.citation: Proc Natl Acad Sci U S A. 2008 Nov 25;105(47):18349-54
references.pmid: 19015532
references.citation: J Biomol NMR. 1995 Sep;6(2):135-40
references.pmid: 8589602
references.citation: J Am Chem Soc. 2007 May 30;129(21):6670-1
references.pmid: 17488074
references.citation: Annu Rev Biophys. 2009;38:385-403
references.pmid: 19245337
references.citation: J Magn Reson. 2003 Nov;165(1):162-74
references.pmid: 14568526
references.citation: Biochim Biophys Acta. 2007 Dec;1768(12):3071-9
references.pmid: 17980349
references.citation: Nat Chem. 2012 Mar 18;4(5):410-7
references.pmid: 22522262
references.citation: J Biomol NMR. 2012 Dec;54(4):325-35
references.pmid: 23180049
references.citation: J Biomol NMR. 2012 Sep;54(1):33-42
references.pmid: 22782234
references.citation: J Mol Biol. 1978 Aug 5;123(2):247-57
references.pmid: 682200
references.citation: Trends Biochem Sci. 2011 Feb;36(2):117-25
references.pmid: 20724162