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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.
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