1H NMR sequential resonance assignments, secondary structure, and global fold in solution of the major (trans-Pro43) form of bovine calbindin D9k.

Kördel J, Forsén S, Chazin WJ
Biochemistry. 1989 28 (17): 7065-74

PMID: 2819050 · DOI:10.1021/bi00443a043

A wide range of two-dimensional 1H NMR experiments have been used to completely assign the 500-MHz 1H NMR spectrum of recombinant Ca2+-saturated bovine calbindin D9k (76 amino acids, Mr = 8500). In solution, calbindin D9k exists as an equilibrium mixture of isoforms with trans (75%) and cis (25%) isomers of the peptide bond at Pro43 [Chazin et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 2195-2198], which results in two sets of 1H NMR signals from approximately half of the amino acids. The complete 1H NMR assignments for the major, trans-Pro43 isoform are presented here. By use of an integrated strategy for spin system identification, 62 of the 76 spin systems could be assigned to the appropriate residue type. Sequence-specific assignments were then obtained by the standard method. Secondary structure elements were identified on the basis of networks of sequential and medium-range nuclear Overhauser effects (NOEs), 3JHN alpha spin coupling constants, and the location of slowly exchanging amide protons. Four helical segments and a short beta-sheet between the two calcium binding loops are found. These elements of secondary structure and a few additional long-range NOEs provide the global fold. Good agreement is found between the solution and crystal structures of the minor A form of bovine calbindin D9k and between the solution structures of the minor A form of bovine calbindin D9k and intact porcine calbindin D9k.

MeSH Terms (12)

Amino Acid Sequence Animals Calbindins Cattle Hydrogen Macromolecular Substances Magnetic Resonance Spectroscopy Models, Structural Molecular Sequence Data Proline Protein Conformation S100 Calcium Binding Protein G

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