title: Secondary structure and side-chain 1H and 13C resonance assignments of calmodulin in solution by heteronuclear multidimensional NMR spectroscopy.
abstract: 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 15N and 13C to a level of greater than 95%. Nearly complete 1H and 13C 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 3JHNH alpha coupling constants. A clear correlation between the 13C 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.
authors.affiliation: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: M
authors.initials: M
authors.lastname: Ikura
authors.forename: S
authors.initials: S
authors.lastname: Spera
authors.forename: G
authors.initials: G
authors.lastname: Barbato
authors.forename: L E
authors.initials: LE
authors.lastname: Kay
authors.forename: M
authors.initials: M
authors.lastname: Krinks
authors.forename: A
authors.initials: A
authors.lastname: Bax
mesh_terms: D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000597:Amino Acids, Branched-Chain / Q000737:chemistry; D000599:Amino Acids, Diamino / Q000737:chemistry; D000600:Amino Acids, Dicarboxylic / Q000737:chemistry; D000818:Animals; D001216:Asparagine / Q000737:chemistry; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry*; D004331:Drosophila melanogaster; D005973:Glutamine / Q000737:chemistry; D005998:Glycine / Q000737:chemistry; D006860:Hydrogen Bonding; D007930:Leucine / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008715:Methionine / Q000737:chemistry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011392:Proline / Q000737:chemistry; D011994:Recombinant Proteins; D012694:Serine / Q000737:chemistry; D012996:Solutions; D013912:Threonine / Q000737:chemistry
chemical_list: D000597:Amino Acids, Branched-Chain; D000599:Amino Acids, Diamino; D000600:Amino Acids, Dicarboxylic; D002147:Calmodulin; D011994:Recombinant Proteins; D012996:Solutions; D005973:Glutamine; D013912:Threonine; D012694:Serine; D001216:Asparagine; D011392:Proline; D008715:Methionine; D007930:Leucine; D000409:Alanine; D002118:Calcium; D005998:Glycine