ArticleAlanine substitutions of noncysteine residues in the cysteine-stabilized   motif |
| Ying-Fang Yang 1 2, Kuo-Chang Cheng 1, Ping-Hsing Tsai 1, Chung-Cheng Liu 2, Tian-Ren Lee 1 *, Ping-Chiang Lyu 1 * |
1Institute of Bioinformatics and Structural Biology, College of Life Sciences, National Tsing-Hua University, Hsin-Chu, Taiwan
2Biomedical Engineering Research Laboratories, Industrial Technology Research Institute, Chu-Tung, Hsin-Chu, Taiwan
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| email: Tian-Ren Lee (trlee@life.nthu.edu.tw) Ping-Chiang Lyu (pclyu@life.nthu.edu.tw) |
*Correspondence to Tian-Ren Lee, Institute of Bioinformatics and Structural Biology, College of Life Sciences, National Tsing-Hua University, 101 Kung-Fu Rd Sec. 2, Hsin-Chu 30013, Taiwan
*Correspondence to Ping-Chiang Lyu, Institute of Bioinformatics and Structural Biology, College of Life Sciences, National Tsing-Hua University, 101 Kung-Fu Rd Sec. 2, Hsin-Chu 30013, Taiwan
Funded by:
National Science Council; Grant Number: NSC 97-2113-M-007-015
plant defensin  -amylase alanine scan circular dichroism spectroscopy fluorescence spectroscopy protein engineering |
The protein scaffold is a peptide framework with a high tolerance of residue modifications. The cysteine-stabilized  motif (CS) consists of an -helix and an antiparallel triple-stranded -sheet connected by two disulfide bridges. Proteins containing this motif share low sequence identity but high structural similarity and 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 CS motif. A systematic alanine substitution is performed on the VrD1. The key residues governing the inhibitory function and structure stability are monitored. Thirty-two of 46 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 CS motif. The information is valuable to the scaffold with the CS motif and protein engineering. |
Received: 1 April 2009; Revised: 4 May 2009; Accepted: 5 May 2009
10.1002/pro.164
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