1H NMR studies of porcine calbindin D9k in solution: sequential resonance assignment, secondary structure, and global fold.
The 1H nuclear magnetic resonance (NMR) spectrum of Ca2+-saturated porcine calbindin D9k (78 amino acids, Mr 8800) has been assigned. Greater than 98% of the 1H resonances, including spin systems for each amino acid residue, have been identified by using an approach that integrates data from a wide range of two-dimensional scalar correlated NMR experiments [Chazin, Rance, & Wright (1988) J. Mol. Biol. 202, 603-626]. Due to the limited quantity of sample and conformational heterogeneity of the protein, two-dimensional nuclear Overhauser effect (NOE) experiments also played an essential role in the identification of spin systems. On the basis of the pattern of scalar connectivities, 43 of the 78 spin systems could be directly assigned to the appropriate residue type. This provided an ample basis for obtaining the sequence-specific resonance assignments. The elements of secondary structure are identified from sequential and medium-range NOEs, values of 3JNH alpha, and the location of slowly exchanging backbone amide protons. Four well-defined helices and a mini beta-sheet between the two calcium binding loops are present in solution. These elements of secondary structure and a few key long-range NOEs provided sufficient information to define the global fold of the protein in solution. Generally good agreement is found between the crystal structure of the minor A form of bovine calbindin D9k and the solution structure of intact porcine calbindin D9k. The only significant difference is a short one-turn helix in the loop between helices II and III in the bovine crystal structure, which is clearly absent in the porcine solution structure.
Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, California 92037.
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Drakenberg
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Hofmann
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Chazin
D000595:Amino Acid Sequence; D000818:Animals; D064026:Calbindins; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011487:Protein Conformation; D064030:S100 Calcium Binding Protein G; D012996:Solutions; D013552:Swine
D064026:Calbindins; D064030:S100 Calcium Binding Protein G; D012996:Solutions