1H NMR sequential resonance assignments, secondary structure, and global fold in solution of the major (trans-Pro43) form of bovine calbindin D9k.
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.
Kördel
J
J
Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, California 92037.
Forsén
S
S
Chazin
W J
WJ
D000595:Amino Acid Sequence; D000818:Animals; D064026:Calbindins; D002417:Cattle; D006859:Hydrogen; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008961:Models, Structural; D008969:Molecular Sequence Data; D011392:Proline; D011487:Protein Conformation; D064030:S100 Calcium Binding Protein G
D064026:Calbindins; D046911:Macromolecular Substances; D064030:S100 Calcium Binding Protein G; D006859:Hydrogen; D011392:Proline