Primary Structure, Sequence-Specific 1H-NMR Assignments and Secondary Structure in Solution of Bromelain Inhibitor VI from Pineapple Stem

Abstract

One of the bromelain inhibitors, isoinhibitor VI (BI-VI), was purified from pineapple stem powder and its complete amino acid sequence was determined by conventional protein sequencing. These results revealed that the protein consists of an 11-residue light chain and a 41-residue heavy chain, cross-linked to each other by disulfide bonds to form the native inhibitor of 52 residues (Mr= 5888). The secondary structure of BI-VI was analyzed based on the sequence-specific 1H resonance assignment of its two-dimensional NMR spectra. BI-VI was shown to be composed of two domains (A and B) which are formed by antiparallel β-sheets, but has no α-helix. These results were consistent with the CD spectra of BI-VI. Residues Lys27-Ile29 (heavy chain) form a triple-stranded antiparallel β-sheet with residues Asp9-Tyr11 and Lys22–Glu24 (heavy chain) in the A domain and residues Cys5–Cys7 (heavy chain) form another triple-stranded β-sheet with residues Cys6–Cys8 (light chain) and Asp32–Ile34 (heavy chain) in the B domain. The secondary structure as well as the primary structure of BI-VI was distinctly different from that of the other cysteine protease inhibitor, cystatin, and from that of basic pancreatic trypsin inhibitor.