| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2000: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1999: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
The propeptide of subtilisin possesses inhibitory activity toward protease in addition to intramolecular chaperone activity that faclitates the folding of subtlisin. The isolated propeptide did not possess tertiary structure. However, successive amino acid replacements Ala47→Phe, Gly13→Ile and Val65→Ile were introduced into the propeptide, the resultant mutants were demonstrated to form defined tertiary structure and to show increased inhibitory activity and resistance toward subtilisin. In this study, intramolecular chaperone activity of the mutated propeptides was examined. The mutated prosubtilisins in which successive amino acid replacements were introduced into the propeptide region were found to refold more rapidly than the wild type, which demonstrates that improvement of intramolecular chaperone activity of the propeptide is closely related to the tertiary structure formation in the propeptide by replacements. Then, the functions of two protease inhibitors, YIB2 and POIA1, whic
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h show sequence similarity to the propeptide were investigated. Although wild-type YIB2 and POIA1 exhibited temporary inhibition toward subtilisin, replacement of C-terminal six residues of these inhibitors by those of the propeptide converted these proteins to more potent and resistant inhibitors of subtilisin. These results suggested that these proteins are unique protease inhibitors which bind the protease using their C-terminal regions in a similar manner to the propeptide, and that their C-terminal residues function as a reactive site toward the protease. On the other hand, possibility of these inhibitors to function as intramolecular chaperone was investigated using fused proteins in which the mutated YIB2 or POIA1 was fused to the N-terminus of the mature region of subtilisin with replacement Ser221→Ala or Cys. The fused proteins were shown to possess secondary structure by their CD spectra after gradual refolding. In addition, autocatalytic cleavage of the peptide bond between the inhibitor and mature regions occurred in the fused proteins with Ser221→Cys replacement in the mature region. These results clearly indicate that the protease inhibitor that is homologous to the propeptide can function as an intramolecular chaperone that accelerates the folding of the mature region, and thus provide new insights in the protease inhibitor and protein folding researches. Less
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