Journal
PROTEIN SCIENCE
Volume 18, Issue 7, Pages 1498-1506Publisher
WILEY
DOI: 10.1002/pro.164
Keywords
plant defensin; alpha-amylase; alanine scan; circular dichroism spectroscopy; fluorescence spectroscopy; protein engineering
Categories
Funding
- National Science Council [NSC 97-2113-M-007-015]
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The protein scaffold is a peptide framework with a high tolerance of residue modifications. The cysteine-stabilized alpha beta motif (CS alpha beta) consists of an alpha-helix and an antiparallel triple-stranded beta-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 alpha beta 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 alpha beta motif. The information is valuable to the scaffold with the CS alpha beta motif and protein engineering.
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