The protein scaffold is a peptide framework with a high tolerance of residue modifications. The cysteine-stabilized ab motif (CSab) consists of an a-helix and an antiparallel triple-stranded b-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 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 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 CSab motif. The information is valuable to the scaffold with the CSab motif and protein engineering.

Plant defensins are a family of small, cysteine-rich and basic proteins. Proteins of the family constitute the major innate immune system of plants and insects. The most significant feature of the family is the conserved cysteine scaffold and one alpha-helix and a triple strand anti parallel beta-sheet connected by the scaffold. Proteins of the family display anti-microbial and anti-fungal activities and a broad spectrum of biological functions. The proteins also demonstrate ultra stable proprieties even in extreme environments. In the past twenty years, patents on the proteins and their applications have increased as technologies have been developed. By utilizing protein engineering approach, medical applications of proteins of the family have been developed. By reviewing patents on proteins of the family, the application development trend of the protein family is traced. Currently, the development of proteins of the family is still in the initial and discovery stage. For their excellent physical and chemical proprieties, it is believed that application of the proteins in the biomedical area should have a high potential and the future of proteins of the plant defensin family.

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