Secondary structure and side-chain sup 1 H and sup 13 C resonance assignments of calmodulin in solution by heteronuclear multidimensional NMR spectroscopy

• Published in: Biochemistry (Easton) 1991-09, Vol.30:38
• Main Authors: Ikura, Mitsuhiko, Spera, S., Barbato, G., Kay, L.E., Bax, A., Krinks, M.
• Format: Article
• Language: English
• Subjects: 550601 - Medicine- Unsealed Radionuclides in Diagnostics; ANIMALS; ARTHROPODS; BARYONS; CALMODULIN; CARBON 13; CARBON ISOTOPES; CHEMICAL SHIFT; DIPTERA; DROSOPHILA; ELEMENTARY PARTICLES; EVEN-ODD NUCLEI; FERMIONS; FLIES; FRUIT FLIES; HADRONS; INSECTS; INVERTEBRATES; ISOTOPES; LIGHT NUCLEI; MAGNETIC RESONANCE; MOLECULAR STRUCTURE; NUCLEAR MAGNETIC RESONANCE; NUCLEI; NUCLEONS; ORGANIC COMPOUNDS; OVERHAUSER EFFECT; PROTEINS; PROTONS; RADIOLOGY AND NUCLEAR MEDICINE; RESONANCE; STABLE ISOTOPES
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00102a013

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 {sup 15}N and {sup 13}C to a level of > 95%. Nearly complete {sup 1}H and {sup 13}C 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 {sup 3}J{sub HNH{alpha}} coupling constants. A clear correlation between the {sup 13}C{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 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.