SECONDARY STRUCTURE AND SIDE-CHAIN H-1 AND C-13 RESONANCE ASSIGNMENTS OF CALMODULIN IN SOLUTION BY HETERONUCLEAR MULTIDIMENSIONAL NMR-SPECTROSCOPY

被引:186
作者
IKURA, M
SPERA, S
BARBATO, G
KAY, LE
KRINKS, M
BAX, A
机构
[1] NIDDKD, CHEM PHYS LAB, BETHESDA, MD 20892 USA
[2] NCI, BIOCHEM LAB, BETHESDA, MD 20892 USA
关键词
D O I
10.1021/bi00102a013
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
Heteronuclear 2D and 3D NMR experiments were carried out on recombinant Drosophila calmodulin (CaM), a protein of 148 residues and with molecular mass of 16.7 kDa, that is uniformly labeled with N-15 and C-13 to a level of > 95%. Nearly complete H-1 and C-13 side-chain assignments for all amino acid residues are obtained by using the 3D HCCH-COSY and HCCH-TOCSY experiments that rely on large heteronuclear one-bond scalar couplings to transfer magnetization and establish through-bond connectivities. The secondary structure of this protein in solution has been elucidated by a qualitative interpretation of nuclear Overhauser effects, hydrogen exchange data, and 3J(HNH-alpha) coupling constants. A clear correlation between the C-13-alpha chemical shift and secondary structure is found. The secondary structure in the two globular domains of Drosophila CaM in solution is essentially identical with that of the X-ray crystal structure of mammalian CaM [Babu, Y., Bugg, C. E., & Cook, W. J. (1988) J. Mol. Biol. 204, 191-204], which consists of two pairs of a "helix-loop-helix" motif in each globular domain. The existence of a short antiparallel beta-sheet between the two loops in each domain has been confirmed. The eight alpha-helix segments identified from the NMR data are located at Glu-6 to Phe-19, Thr-29 to Ser-38, Glu-45 to Glu-54, Phe-65 to Lys-77, Glu-82 to Asp-93, Ala-102 to Asn-111, Asp-118 to Glu-127, and Tyr-138 to Thr-146. Although the crystal structure has a long "central helix" from Phe-65 to Phe-92 that connects the two globular domains, NMR data indicate that residues Asp-78 to Ser-81 of this central helix adopt a nonhelical conformation with considerable flexibility.
引用
收藏
页码:9216 / 9228
页数:13
相关论文
共 64 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]   STRUCTURE OF CALMODULIN REFINED AT 2.2 A RESOLUTION [J].
BABU, YS ;
BUGG, CE ;
COOK, WJ .
JOURNAL OF MOLECULAR BIOLOGY, 1988, 204 (01) :191-204
[3]  
BALDISSERI DM, 1991, IN PRESS FEBS LETT
[4]   CORRELATION OF PROTON AND N-15 CHEMICAL-SHIFTS BY MULTIPLE QUANTUM NMR [J].
BAX, A ;
GRIFFEY, RH ;
HAWKINS, BL .
JOURNAL OF MAGNETIC RESONANCE, 1983, 55 (02) :301-315
[5]   REMOVAL OF F1-BASE-LINE DISTORTION AND OPTIMIZATION OF FOLDING IN MULTIDIMENSIONAL NMR-SPECTRA [J].
BAX, A ;
IKURA, M ;
KAY, LE ;
ZHU, G .
JOURNAL OF MAGNETIC RESONANCE, 1991, 91 (01) :174-178
[6]   H-1-H-1 CORRELATION VIA ISOTROPIC MIXING OF C-13 MAGNETIZATION, A NEW 3-DIMENSIONAL APPROACH FOR ASSIGNING H-1 AND C-13 SPECTRA OF C-13-ENRICHED PROTEINS [J].
BAX, A ;
CLORE, GM ;
GRONENBORN, AM .
JOURNAL OF MAGNETIC RESONANCE, 1990, 88 (02) :425-431
[7]   PRACTICAL ASPECTS OF PROTON CARBON CARBON PROTON 3-DIMENSIONAL CORRELATION SPECTROSCOPY OF C-13-LABELED PROTEINS [J].
BAX, A ;
CLORE, GM ;
DRISCOLL, PC ;
GRONENBORN, AM ;
IKURA, M ;
KAY, LE .
JOURNAL OF MAGNETIC RESONANCE, 1990, 87 (03) :620-627
[8]   REEXAMINATION OF A SEQUENCE DESIGNED TO CANCEL C-13 SIGNALS OF PROTONATED CARBONS [J].
BENDALL, MR ;
PEGG, DT .
JOURNAL OF MAGNETIC RESONANCE, 1983, 52 (01) :136-138
[9]   PEPTIDE GROUP SHIFTS [J].
CLAYDEN, NJ ;
WILLIAMS, RJP .
JOURNAL OF MAGNETIC RESONANCE, 1982, 49 (03) :383-396
[10]   IDENTIFICATION AND LOCALIZATION OF BOUND INTERNAL WATER IN THE SOLUTION STRUCTURE OF INTERLEUKIN-1-BETA BY HETERONUCLEAR 3-DIMENSIONAL H-1 ROTATING-FRAME OVERHAUSER N-15-H-1 MULTIPLE QUANTUM COHERENCE NMR-SPECTROSCOPY [J].
CLORE, GM ;
BAX, A ;
WINGFIELD, PT ;
GRONENBORN, AM .
BIOCHEMISTRY, 1990, 29 (24) :5671-5676