title: Lipid bilayer-bound conformation of an integral membrane beta barrel protein by multidimensional MAS NMR.
abstract: The human voltage dependent anion channel 1 (VDAC) is a 32 kDa β-barrel integral membrane protein that controls the transport of ions across the outer mitochondrial membrane. Despite the determination of VDAC solution and diffraction structures, a structural basis for the mechanism of its function is not yet fully understood. Biophysical studies suggest VDAC requires a lipid bilayer to achieve full function, motivating the need for atomic resolution structural information of VDAC in a membrane environment. Here we report an essential step toward that goal: extensive assignments of backbone and side chain resonances for VDAC in DMPC lipid bilayers via magic angle spinning nuclear magnetic resonance (MAS NMR). VDAC reconstituted into DMPC lipid bilayers spontaneously forms two-dimensional lipid crystals, showing remarkable spectral resolution (0.5-0.3 ppm for (13)C line widths and <0.5 ppm (15)N line widths at 750 MHz). In addition to the benefits of working in a lipid bilayer, several distinct advantages are observed with the lipid crystalline preparation. First, the strong signals and sharp line widths facilitated extensive NMR resonance assignments for an integral membrane β-barrel protein in lipid bilayers by MAS NMR. Second, a large number of residues in loop regions were readily observed and assigned, which can be challenging in detergent-solubilized membrane proteins where loop regions are often not detected due to line broadening from conformational exchange. Third, complete backbone and side chain chemical shift assignments could be obtained for the first 25 residues, which comprise the functionally important N-terminus. The reported assignments allow us to compare predicted torsion angles for VDAC prepared in DMPC 2D lipid crystals, DMPC liposomes, and LDAO-solubilized samples to address the possible effects of the membrane mimetic environment on the conformation of the protein. Concluding, we discuss the strengths and weaknesses of the reported assignment approach and the great potential for even more complete assignment studies and de novo structure determination via (1)H detected MAS NMR.
authors: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
authors: Matthew T
authors: MT
authors: Eddy
authors: Yongchao
authors: Y
authors: Su
authors: Robert
authors: R
authors: Silvers
authors: Loren
authors: L
authors: Andreas
authors: Lindsay
authors: L
authors: Clark
authors: Gerhard
authors: G
authors: Wagner
authors: Guido
authors: G
authors: Pintacuda
authors: Lyndon
authors: L
authors: Emsley
authors: Robert G
authors: RG
authors: Griffin
mesh_terms: D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry*; D008279:Magnetic Resonance Imaging; D008823:Micelles; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D050995:Voltage-Dependent Anion Channel 1 / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*
chemical_list: D008051:Lipid Bilayers; D008823:Micelles; C494762:VDAC1 protein, human; D050995:Voltage-Dependent Anion Channel 1
references: J Am Chem Soc. 2012 Apr 11;134(14):6375-87
references: 22435461
references: Proc Natl Acad Sci U S A. 2012 Sep 18;109(38):15265-70
references: 22942391
references: J Biomol NMR. 2012 Nov;54(3):291-305
references: 22986689
references: Nature. 2012 Nov 29;491(7426):779-83
references: 23086146
references: Science. 2013 Mar 29;339(6127):1600-4
references: 23539602
references: Nat Methods. 2013 Oct;10(10):1007-12
references: 24013819
references: J Magn Reson. 2014 Feb;239:100-9
references: 24412099
references: Solid State Nucl Magn Reson. 2014 Jul-Sep;61-62:1-7
references: 24837131
references: J Am Chem Soc. 2014 Sep 3;136(35):12489-97
references: 25102442
references: Nature. 2014 Dec 11;516(7530):207-12
references: 25383531
references: Biochemistry. 2000 Jan 18;39(2):362-71
references: 10630997
references: Proc Natl Acad Sci U S A. 2001 Feb 27;98(5):2358-63
references: 11226244
references: Nat Struct Biol. 2001 Apr;8(4):334-8
references: 11276254
references: J Am Chem Soc. 2002 Sep 11;124(36):10728-42
references: 12207528
references: Proc Natl Acad Sci U S A. 2002 Oct 15;99(21):13560-5
references: 12357033
references: J Mol Biol. 2004 Mar 5;336(5):1211-21
references: 15037080
references: J Am Chem Soc. 2004 Jun 16;126(23):7196-7
references: 15186155
references: Curr Opin Struct Biol. 2004 Oct;14(5):554-61
references: 15465315
references: J Membr Biol. 1976 Dec 28;30(2):99-120
references: 1011248
references: Biochemistry. 1985 Nov 19;24(24):6955-62
references: 4074732
references: Biochemistry. 1992 Sep 1;31(34):7931-8
references: 1510979
references: J Am Chem Soc. 2008 Jan 16;130(2):408-9
references: 18092784
references: Proc Natl Acad Sci U S A. 2008 Jan 22;105(3):883-8
references: 18195364
references: Protein Sci. 2008 Feb;17(2):199-204
references: 18227427
references: Nature. 2008 Jan 31;451(7178):591-5
references: 18235503
references: J Biomol NMR. 1995 Nov;6(3):277-93
references: 8520220
references: J Biol Chem. 1996 Nov 8;271(45):28006-8
references: 8910409
references: J Membr Biol. 1997 Jun 1;157(3):271-9
references: 9178614
references: J Bioenerg Biomembr. 1997 Dec;29(6):525-31
references: 9559853
references: Biochemistry. 1998 Jun 2;37(22):8088-96
references: 9609703
references: Biochemistry. 1998 Jul 28;37(30):10854-9
references: 9692976
references: J Struct Biol. 1999 Aug;127(1):64-71
references: 10479618
references: Proc Natl Acad Sci U S A. 2005 Aug 2;102(31):10870-5
references: 16043693
references: Chembiochem. 2005 Sep;6(9):1679-84
references: 16138308
references: J Am Chem Soc. 2005 Sep 21;127(37):12965-74
references: 16159291
references: Proc Natl Acad Sci U S A. 2005 Nov 1;102(44):15871-6
references: 16247008
references: J Biomol NMR. 2006 Sep;36(1):55-71
references: 16964530
references: J Recept Signal Transduct Res. 2006;26(5-6):379-93
references: 17118788
references: Angew Chem Int Ed Engl. 2007;46(3):459-62
references: 17001715
references: Biochemistry. 2007 Jan 16;46(2):514-25
references: 17209561
references: J Mol Biol. 2007 Apr 20;368(1):66-78
references: 17336328
references: Proc Natl Acad Sci U S A. 2007 Oct 9;104(41):16140-5
references: 17911261
references: Proc Natl Acad Sci U S A. 2007 Nov 13;104(46):18031-6
references: 17989232
references: Science. 2007 Nov 23;318(5854):1266-73
references: 17962519
references: Science. 2007 Nov 23;318(5854):1258-65
references: 17962520
references: Biochim Biophys Acta. 2007 Dec;1774(12):1604-13
references: 17974509
references: J Biomol NMR. 2008 May;41(1):9-15
references: 18404253
references: Nat Struct Mol Biol. 2008 Jun;15(6):605-12
references: 18488040
references: J Am Chem Soc. 2008 Jun 11;130(23):7427-35
references: 18479093
references: Science. 2008 Aug 29;321(5893):1206-10
references: 18755977
references: Proc Natl Acad Sci U S A. 2008 Oct 7;105(40):15370-5
references: 18832158
references: Proc Natl Acad Sci U S A. 2008 Nov 18;105(46):17742-7
references: 18988731
references: J Magn Reson. 2009 Jan;196(1):84-7
references: 18930418
references: J Mol Biol. 2009 Jan 9;385(1):117-30
references: 18952100
references: Nat Struct Mol Biol. 2009 Feb;16(2):168-75
references: 19182802
references: J Mol Biol. 2009 Mar 6;386(4):1078-93
references: 19244620
references: Annu Rev Biophys. 2009;38:385-403
references: 19245337
references: Proc Natl Acad Sci U S A. 2009 Jun 9;106(23):9244-9
references: 19474298
references: J Mol Biol. 2009 Jul 24;390(4):662-71
references: 19447114
references: J Biomol NMR. 2009 Aug;44(4):213-23
references: 19548092
references: J Biomol NMR. 2009 Aug;44(4):245-60
references: 19609683
references: Biochim Biophys Acta. 2009 Dec;1788(12):2563-74
references: 19799854
references: Angew Chem Int Ed Engl. 2010 Mar 1;49(10):1882-5
references: 20140924
references: Biomol NMR Assign. 2010 Apr;4(1):29-32
references: 20437141
references: J Mol Biol. 2010 Aug 13;401(2):155-66
references: 20600123
references: J Am Chem Soc. 2010 Aug 18;132(32):10958-60
references: 20698642
references: Proc Natl Acad Sci U S A. 2011 May 31;108(22):9101-6
references: 21576492
references: Nature. 2011 Jul 7;475(7354):65-70
references: 21697825
references: Structure. 2011 Sep 7;19(9):1283-93
references: 21885291
references: J Biomol NMR. 2011 Nov;51(3):227-33
references: 21938394
references: Angew Chem Int Ed Engl. 2011 Dec 9;50(50):11942-6
references: 22034093
references: Nature. 2012 Feb 9;482(7384):237-40
references: 22286059
references: Annu Rev Phys Chem. 2012;63:1-24
references: 22136620