A sensitive and robust method for obtaining intermolecular NOEs between side chains in large protein complexes

被引:59
作者
Gross, JD [1 ]
Gelev, VM [1 ]
Wagner, G [1 ]
机构
[1] Harvard Univ, Sch Med, Dept Biol Chem & Mol Pharmacol, Boston, MA 02115 USA
关键词
biosynthetic precursors; C-13-methyl; intermolecular NOEs; isotope labeling of proteins; protein complex;
D O I
10.1023/A:1022890112109
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
A method for measuring intermolecular NOEs in protein complexes based on asymmetric sample deuteration is described. C-13/H-1-I,L,V-methyl, U-H-2 labeled protein is produced using the biosynthetic precursors [gamma-C-13]- alpha-ketobutyrate and [gamma,gamma'-C-13(2)]-alpha-ketoisovalerate. The labeled protein is mixed with its unlabeled binding partner and a 3D C-13-HMQC-NOESY is recorded, yielding unambiguous intermolecular aromatic/methyl NOEs. A simple synthesis of the biosynthetic precursors via reaction of diethyl oxalate with alkyl Grignard compounds is reported. The method is demonstrated for a 35 kDa heterodimeric protein complex dissolved in a CHAPS micelle. This approach will facilitate the solution structure determination of protein/protein, protein/ligand or protein/nucleic acid complexes.
引用
收藏
页码:235 / 242
页数:8
相关论文
共 38 条
[1]   SEQUENCE-SPECIFIC H-1-NMR ASSIGNMENTS AND SECONDARY STRUCTURE IN SOLUTION OF ESCHERICHIA-COLI TRP REPRESSOR [J].
ARROWSMITH, CH ;
PACHTER, R ;
ALTMAN, RB ;
IYER, SB ;
JARDETZKY, O .
BIOCHEMISTRY, 1990, 29 (27) :6332-6341
[2]  
BATTISTE JL, 2003, BIOL MAGN RESON, V20, P79
[3]   Solution structure of paramagnetic metalloproteins [J].
Bertini, I ;
Rosato, A ;
Turano, P .
PURE AND APPLIED CHEMISTRY, 1999, 71 (09) :1717-1725
[4]   THERMAL REARRANGEMENTS OF ALLENES - SYNTHESIS AND MECHANISM OF CYCLOAROMATIZATION OF PI-BRIDGED AND HETEROATOM BRIDGED DIALLENES [J].
BRAVERMAN, S ;
DUAR, Y .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 1990, 112 (15) :5830-5837
[5]   SUPPRESSION OF CROSS-RELAXATION EFFECTS IN TOCSY SPECTRA VIA A MODIFIED DIPSI-2 MIXING SEQUENCE [J].
CAVANAGH, J ;
RANCE, M .
JOURNAL OF MAGNETIC RESONANCE, 1992, 96 (03) :670-678
[6]   SYNTHESIS OF 4,4-DIMETHYL-DIHYDRO-2,3-FURANEDIONE [J].
FIZET, C .
HELVETICA CHIMICA ACTA, 1982, 65 (07) :2024-2028
[7]   Production and incorporation of N-15, C-13, H-2 (H-1-delta 1 methyl) isoleucine into proteins for multidimensional NMR studies [J].
Gardner, KH ;
Kay, LE .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 1997, 119 (32) :7599-7600
[8]   Global folds of highly deuterated, methyl-protonated proteins by multidimensional NMR [J].
Gardner, KH ;
Rosen, MK ;
Kay, LE .
BIOCHEMISTRY, 1997, 36 (06) :1389-1401
[9]   An (H)C(CO)NH-TOCSY pulse scheme for sequential assignment of protonated methyl groups in otherwise deuterated N-15,C-13-labeled proteins [J].
Gardner, KH ;
Konrat, R ;
Rosen, MK ;
Kay, LE .
JOURNAL OF BIOMOLECULAR NMR, 1996, 8 (03) :351-356
[10]   CORRELATION OF BACKBONE AMIDE AND ALIPHATIC SIDE-CHAIN RESONANCES IN C-13/N-15-ENRICHED PROTEINS BY ISOTROPIC MIXING OF C-13 MAGNETIZATION [J].
GRZESIEK, S ;
ANGLISTER, J ;
BAX, A .
JOURNAL OF MAGNETIC RESONANCE SERIES B, 1993, 101 (01) :114-119