Domain analysis of tarocystatin by bioimformatic and biochemical assay

• Main Authors: Ming-Hung Chu, 朱明宏
• Other Authors: Yi-Sheng Cheng
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/77037059083686774118

碩士 === 國立臺灣大學 === 植物科學研究所 === 97 === Tarocystatin is a member of group-2 phytocystatin which could help plants against insect and pathogen attack. It shares a similar cystatin domain in the N-terminal region (NtD), and contained an extended C-terminal domain (CtD). For identifying the interaction of NtD and CtD, the glutaraldehyde cross-linking and yeast two-hybrid assays were carried out, the results showed a weak interaction between NtD and CtD, and tarocystatin could form homodimer by interaction of NtD and NtD. According to amino acids sequence alignment and secondary structure prediction, the fold of CtD should be a cystatin-like domain (CY-L domain) with similar folding to NtD which is 1 α-helix followed by 4 β-sheets. Based on domain interaction and bioinformatic analyses, we performed homology modeling and protein-protein docking to build the two models of tarocystatin using OC-I as the template. Moreover, we confirmed that CtD possess weak papain activitation property by in-gel inhibitory activity assay, full-length of tarocystatin showed higher anti-papain activity than NtD, and the GST fused tarocystatin augment their original property than none fused protein. As the results, we speculated that native tarocystatin could exist both monomeric and homodimeric form, and CtD might play a role in enhancing the inhibitory activity of tarocystatin.