1H-NMR studies of the structure and stability of the bovine pancreatic secretory trypsin inhibitor.

The tertiary structure of the isoinhibitor A (Kazal) isolated from bovine pancreatic tissue has been characterized by 1H-NMR studies in 2H2O solution. A number of slowly exchanging backbone amides are observed. The dynamics of the aromatic side chains and their interaction with methyl groups from aliphatic residues are essentially identical in the bovine and in the homologous inhibitor from porcine pancreas. Both inhibitors contain two tyrosines in positions 20 and 31 as the only aromatic residues. The previous assignment of the aromatic resonances in the porcine inhibitor is valid for the bovine homolog, as deduced from selective nuclear Overhauser enhancement (NOE) experiments. The tyrosine which inhibits an NMR spectral pattern characteristic of rapid ring motion does not show appreciable NOE to backbone or sidechain protons, in agreement with the solvent exposure predicted for Tyr-20 in the porcine inhibitor. In contrast, the immobilized Tyr-31 exhibits a NOE pattern that indicates close interaction with a number of backbone amide protons and aliphatic side chain groups, confirming that the aromatic ring is buried. At 90 degrees C, pH 5, the protein is still substantially folded, as manifested by both backbone and side chain resonances. The results are compared with previous optical rotatory dispersion findings. A number of side chain resonances were assigned to specific amino acid residues in the sequence.