Site-Directed Mutagenesis Studies of Potential Structural Element of Vigna radiata Plant Defensin 1 Involved in Inhibiting Insect α-Amylase

• Main Authors: Kuo-Chang Cheng, 鄭國璋
• Other Authors: Ping-Chiang Lyu
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/17595630855887881467

碩士 === 國立清華大學 === 生物資訊與結構生物研究所 === 95 === Vigna radiata plant defensin 1 (VrD1) is the first reported plant defensin exhibiting insecticidal activity against Callosobruchus chinensis (bruchid). Three dimensional structure of VrD1 has been determined by nuclear magnetic resonance spectroscopy in our laboratory. Nevertheless, the mechanism of insecticidal activity is unclear. According to our previous work, VrD1 has shown to inhibit Tenebrio molitor α-amylase (TMA) in vitro which may trigger insecticidal activity. Computational docking model of VrD1-TMA complex also implied that loop L3 of VrD1 is important for this inhibition. Among plant defensins of known structure, VrD1 is the first case containing a 310 helix. Based on protein sequence alignments, VrD1 is different from other defensins and contains an arginine in place of glutamate at the residue 26. We propose that this residue may induce a shift in the orientation of Trp10, thereby facilitating the 310 helix formation and then contributing to the stability of global structure. Therefore, this study would focuse on the influence of loop L3 and 310 helix of VrD1 on its inhibitory function. Site-directed mutagenesis was carried out to study critical residues of VrD1 involving in TMA inhibitory activity. The secondary structures of purified proteins were examined by circular dichroism (CD). Our results showed that several mutants significantly decreased in TMA inhibition, indicating that the loop L3 and 310 helix indeed play important roles in VrD1 insecticidal function.