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Alanine substitutions of noncysteine residues in the cysteine‐stabilized αβ motif
Author(s) -
Yang YingFang,
Cheng KuoChang,
Tsai PingHsing,
Liu ChungCheng,
Lee TianRen
Publication year - 2009
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.164
Subject(s) - cysteine , circular dichroism , antiparallel (mathematics) , alanine , protein engineering , chemistry , alanine scanning , peptide , structural motif , mutant , peptide sequence , defensin , protein structure , amino acid , protein folding , biochemistry , protein secondary structure , protein design , site directed mutagenesis , stereochemistry , mutagenesis , enzyme , gene , physics , quantum mechanics , magnetic field
The protein scaffold is a peptide framework with a high tolerance of residue modifications. The cysteine‐stabilized αβ motif (CSαβ) consists of an α‐helix and an antiparallel triple‐stranded β‐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 CSαβ 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 CSαβ motif. The information is valuable to the scaffold with the CSαβ motif and protein engineering.