Study on Functional Domain of Tarocystatin for Oligomerization and Inhibition Activity

• Main Authors: Kai-Wen Fan, 范凱雯
• Other Authors: 鄭貽生
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/95215742628522791730

碩士 === 國立臺灣大學 === 植物科學研究所 === 100 === Tarocystatin, a member of group-2 phytocystatin, contains a conserved N-terminal cystatin domain (NtD) and a C-terminal cystatin-like extension (CtE). It has been reported to act as defense proteins against pathogens. Melting temperature (Tm) of tarocystatin full-length (FL), NtD and CtE were measured using differential scanning calorimetry. Their Tm was higher than 75ºC and suggested they should have high thermal stability. Subcellular localization suggested the existence of tarocystatin in both the cytoplasm and the nucleus. Secondary structure analysis from circular dichroism spectroscopy revealed that FL and CtE have similar conformation to that of NtD with mainly composed of β-strands. Gel filtration chromatography combining with isothermal titration calorimetry (ITC) method indicated that tarocystatin might exist monomer and oligomer at ambient pH, and homodimeric FL could form by domain-domain interaction, especially by NtD and NtD, but not by NtD and CtE. In addition, ITC results also showed that monomeric FL and NtD can bind to papain with high binding affinity, in contrast with the dimeric FL, NtD and monomeric CtE did not show any binding affinity to papain. Inhibition activity assay revealed that NtD possess higher anti-papain activity than FL, but CtE could slightly increase papain activity, and FL and NtD both were unstable at > 70ºC. Thus, we propose that the presence of oligomerization of tarocystatin be relevant for inhibitory regulation in vivo and CtE provide inhibitory variety of tarocystatin in cystatin protein evolution.