The protein scaffold is a peptide framework with a high tolerance of residue modifications. The cysteine-stabilized alpha beta□motif (CSab□□) consists of an alpha-helix and an anti-parallel triple-stranded beta-sheet connected by two pairs of disulfide bridges. The protein containing the motif share low sequence identity but high structural similarity, and it has been suggested as a good scaffold for protein engineering. The Vigna radiate defensin 1 (VrD1), a plant defensin, serves here as a model protein to probe the amino acid tolerance of CSab motif. A systematic alanine substitution is performed on the VrD1. The key residues governing the inhibitory function and structure stability are monitored. Thirty-two out of forty-six residue positions of VrD1 are altered by site-directed mutagenesis techniques. The circular dichroism spectrum, intrinsic fluorescence spectrum and chemical denaturation are used to analyze the conformation and structural stability of proteins. The secondary structures were highly tolerant to the amino acid substitutions; however the protein stabilities were varied for each mutant. Many mutants, although they maintained their conformations, altered their inhibitory function significantly. In this study, we reported the first alanine scan on the plant defensin containing the CSab motif. The information is valuable to the scaffold with the CSab motif and protein engineering.

蛋白質鷹架為一對氨基酸置換具有高耐受度的胜肽骨架，本研究針對具有cysteine-stabilized □□ motif（CS□□ motif）之蛋白質鷹架進行系統性的耐受度研究。具有此一結構的蛋白質族群，其氨基酸序列相似度低，但可摺疊成相似度極高的立體構造，一般認為適合應用於蛋白質工程改質研究。綠豆第一型防禦素為一具有CSab motif的蛋白質，本研究中應用此一蛋白質做為模型，探討CSab motif對於氨基酸置換之耐受性，利用系統性丙氨酸置換方法，搜索對CS□□ motif之結構穩定性及生化功能具有決定性影響的氨基酸位置。本研究以核苷酸聚合酶鏈鎖反應方法對模型蛋進行點突變，對各突變蛋白進行旋光光譜、內生性螢光光譜及化學變性之測量，以酵素抑制能力作為生化指標。結果顯示，雖然部份突變完全失去酵素抑制能力，但所有突變蛋白質的結構穩定性並未有顯著變化。本研究為第一個對CSab motif進行完整的丙氨酸置換研究，所得之結果對CSab motif之蛋白質工程研究具有高度的應用參考價值。

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