Assignment strategies for large proteins by magic-angle spinning NMR: the 21-kDa disulfide-bond-forming enzyme DsbA.
We present strategies for chemical shift assignments of large proteins by magic-angle spinning solid-state NMR, using the 21-kDa disulfide-bond-forming enzyme DsbA as prototype. Previous studies have demonstrated that complete de novo assignments are possible for proteins up to approximately 17 kDa, and partial assignments have been performed for several larger proteins. Here we show that combinations of isotopic labeling strategies, high field correlation spectroscopy, and three-dimensional (3D) and four-dimensional (4D) backbone correlation experiments yield highly confident assignments for more than 90% of backbone resonances in DsbA. Samples were prepared as nanocrystalline precipitates by a dialysis procedure, resulting in heterogeneous linewidths below 0.2 ppm. Thus, high magnetic fields, selective decoupling pulse sequences, and sparse isotopic labeling all improved spectral resolution. Assignments by amino acid type were facilitated by particular combinations of pulse sequences and isotopic labeling; for example, transferred echo double resonance experiments enhanced sensitivity for Pro and Gly residues; [2-(13)C]glycerol labeling clarified Val, Ile, and Leu assignments; in-phase anti-phase correlation spectra enabled interpretation of otherwise crowded Glx/Asx side-chain regions; and 3D NCACX experiments on [2-(13)C]glycerol samples provided unique sets of aromatic (Phe, Tyr, and Trp) correlations. Together with high-sensitivity CANCOCA 4D experiments and CANCOCX 3D experiments, unambiguous backbone walks could be performed throughout the majority of the sequence. At 189 residues, DsbA represents the largest monomeric unit for which essentially complete solid-state NMR assignments have so far been achieved. These results will facilitate studies of nanocrystalline DsbA structure and dynamics and will enable analysis of its 41-kDa covalent complex with the membrane protein DsbB, for which we demonstrate a high-resolution two-dimensional (13)C-(13)C spectrum.
Sperling
Lindsay J
LJ
Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA.
Berthold
Deborah A
DA
Sasser
Terry L
TL
Jeisy-Scott
Victoria
V
Rienstra
Chad M
CM
D003460:Crystallization; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D007554:Isotopes / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D019704:Protein Disulfide-Isomerases / Q000737:chemistry*; D013194:Staining and Labeling / Q000379:methods
D029968:Escherichia coli Proteins; D007554:Isotopes; D019704:Protein Disulfide-Isomerases; C526648:dsbA protein, E coli
J Biol Chem. 2009 Apr 10;284(15):10150-9
19181668
Cell. 1991 Nov 1;67(3):581-9
1934062
Angew Chem Int Ed Engl. 2007;46(3):459-62
17001715
J Biol Chem. 2007 Apr 6;282(14):10263-71
17267399
J Biomol NMR. 2007 Nov;39(3):197-211
17899395
J Biol Chem. 2004 Mar 26;279(13):12967-73
14726535
J Magn Reson. 2003 Jun;162(2):479-86
12810033
J Am Chem Soc. 2004 Apr 28;126(16):5323-31
15099118
J Mol Biol. 2009 Mar 6;386(4):1078-93
19244620
Biochemistry. 1993 May 18;32(19):5083-92
8494885
Protein Sci. 1998 May;7(5):1233-44
9605329
J Am Chem Soc. 2002 Sep 11;124(36):10728-42
12207528
J Am Chem Soc. 2004 Jun 2;126(21):6720-7
15161300
FEMS Microbiol Rev. 2007 Sep;31(5):626-36
17696887
J Am Chem Soc. 2009 Jan 28;131(3):985-92
19123862
J Biomol NMR. 1999 Mar;13(3):289-302
10212987
Science. 2008 Mar 14;319(5869):1523-6
18339938
Nat Rev Microbiol. 2009 Mar;7(3):215-25
19198617
J Mol Biol. 1993 Apr 5;230(3):1097-100
8478925
Proc Natl Acad Sci U S A. 2008 Mar 25;105(12):4621-6
18344321
J Magn Reson. 2006 Apr;179(2):206-16
16406627
Annu Rev Biochem. 2003;72:111-35
12524212
Magn Reson Chem. 2007 Dec;45 Suppl 1:S84-92
18157839
Biochim Biophys Acta. 2008 Sep;1777(9):1098-108
18267103
FEBS Lett. 2008 Oct 15;582(23-24):3301-7
18775700
Angew Chem Int Ed Engl. 2007;46(7):1079-82
17191298
J Biol Chem. 2004 Feb 20;279(8):6761-8
14634016
J Am Chem Soc. 2004 Dec 22;126(50):16608-20
15600367
Biochim Biophys Acta. 2008 Apr;1783(4):520-9
18082634
Biochemistry. 1998 May 5;37(18):6263-76
9572841
J Phys Chem B. 2007 Dec 27;111(51):14362-9
18052145
Protein Sci. 1998 Oct;7(10):2065-80
9792093
Fold Des. 1998;3(3):161-71
9562546
J Am Chem Soc. 2004 Jun 16;126(23):7196-7
15186155
Nature. 1993 Sep 30;365(6445):464-8
8413591
EMBO J. 2002 Jun 3;21(11):2646-54
12032077
Acta Biochim Pol. 2006;53(3):585-9
17019443
J Mol Biol. 2009 Feb 6;385(5):1481-97
19041879
Protein Sci. 2008 Feb;17(2):199-204
18227427
J Biomol NMR. 2000 Mar;16(3):209-19
10805127
J Biomol NMR. 2007 Oct;39(2):107-31
17687624
Int J Biochem Cell Biol. 2006;38(7):1050-62
16446111
Cell. 1995 Dec 15;83(6):947-55
8521518
J Biomol NMR. 1995 Nov;6(3):277-93
8520220
Mol Cell. 2008 Sep 26;31(6):896-908
18922471
J Magn Reson. 2011 Dec;213(2):413-7
22152360
J Biomol NMR. 2003 Dec;27(4):323-39
14512730
Chembiochem. 2007 Mar 26;8(5):486-9
17300109
Nature. 2002 Nov 7;420(6911):98-102
12422222
Biochemistry. 2003 Oct 7;42(39):11476-83
14516199
Annu Rev Phys Chem. 2002;53:349-78
11972012
Structure. 2006 Nov;14(11):1701-10
17098195
Nature. 2006 Apr 13;440(7086):959-62
16612389
Science. 2004 Jan 23;303(5657):534-7
14739460
J Biomol NMR. 2009 Aug;44(4):245-60
19609683
Cell. 2006 Nov 17;127(4):789-801
17110337
J Am Chem Soc. 2005 Sep 7;127(35):12291-305
16131207
EMBO J. 2009 Mar 18;28(6):779-91
19214188
Chembiochem. 2007 Mar 5;8(4):434-42
17285659
Proc Natl Acad Sci U S A. 2008 Aug 19;105(33):11933-8
18695247
J Biomol NMR. 1994 Mar;4(2):171-80
8019132
J Phys Chem B. 2005 Sep 29;109(38):18135-45
16853329
J Am Chem Soc. 2008 Mar 19;130(11):3579-89
18284240
J Magn Reson. 1999 Aug;139(2):389-401
10423377
Chembiochem. 2001 Apr 2;2(4):272-81
11828455
J Am Chem Soc. 2008 Mar 26;130(12):3959-66
18321098
J Am Chem Soc. 2006 Aug 9;128(31):9992-3
16881610
J Biol Chem. 2009 Jun 26;284(26):17835-45
19389711
Angew Chem Int Ed Engl. 2005 Mar 29;44(14):2089-92
15744789
Proc Natl Acad Sci U S A. 2008 Nov 11;105(45):17284-9
18988744
J Biomol NMR. 2001 May;20(1):71-5
11430757
J Phys Chem B. 2006 Jun 8;110(22):10926-36
16771346
J Am Chem Soc. 2007 May 2;129(17):5318-9
17425317
Protein Expr Purif. 2005 May;41(1):207-34
15915565
Structure. 1998 Jun 15;6(6):757-67
9655827