Alanine substitutions of noncysteine residues in the cysteine-stabilized alphabeta motif.
The protein scaffold is a peptide framework with a high tolerance of residue modifications. The cysteine-stabilized alphabeta motif (CS alphabeta) 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 alphabeta 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 alphabeta motif. The information is valuable to the scaffold with the CS alphabeta motif and protein engineering.
Yang
Ying-Fang
YF
Institute of Bioinformatics and Structural Biology, College of Life Sciences, National Tsing-Hua University, Hsin-Chu, Taiwan.
Cheng
Kuo-Chang
KC
Tsai
Ping-Hsing
PH
Liu
Chung-Cheng
CC
Lee
Tian-Ren
TR
Lyu
Ping-Chiang
PC
D000409:Alanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000502:physiology*; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D023082:Defensins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007887:Fabaceae / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011489:Protein Denaturation; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D055550:Protein Stability; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence; D000516:alpha-Amylases / Q000378:metabolism
D023082:Defensins; D010940:Plant Proteins; D011506:Proteins; D000516:alpha-Amylases; D003545:Cysteine; D000409:Alanine