Structure-function validation of high lysine analogs of alpha-hordothionin designed by protein modeling.
Cereal grains and legume seeds, which are key protein sources for the vegetarian diet, are generally deficient in essential amino acids. Maize, in particular, is deficient in lysine. The inherent lack of lysine-rich proteins in maize has necessitated the search for heterologous proteins enriched in this amino acid, the isolation of the corresponding gene and its ultimate introduction into maize through plant transformation techniques. However, a rate-limiting step to this strategy has been the availability of plant-derived lysine-rich proteins. An appealing solution to the problem is to artificially increase the lysine content of a given protein by mutating appropriate residues to lysine. Here, we expound this strategy, starting with the protein alpha-hordothionin that is derived from barley seeds and consists of five lysine residues in a total of 45 amino acids (11% lysine). To facilitate rational substitutions, the 3-D structure of the protein has been determined by homology modeling with crambin. Based on this model, we have identified surface residues amenable to substitution with lysine. Furthermore, the acceptability of the mutations has been validated through the synthesis and characterization of the derivatives. To this end, our approach has permitted the creation of a modified alpha-hordothionin protein that has a lysine content of approximately 27% and retains the antifungal activity of the wild-type protein.
Rao
A G
AG
Department of Traits and Technology Development, Pioneer Hi-Bred International Inc., Johnston, IA 50131-1004, USA.
Hassan
M
M
Hempel
J C
JC
D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000737:chemistry; D023181:Antimicrobial Cationic Peptides; D004220:Disulfides / Q000737:chemistry; D001467:Hordeum / Q000737:chemistry*; D008239:Lysine / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics; D015202:Protein Engineering; D017510:Protein Folding; D012639:Seeds / Q000737:chemistry*; D013329:Structure-Activity Relationship
D000935:Antifungal Agents; D023181:Antimicrobial Cationic Peptides; D004220:Disulfides; D010940:Plant Proteins; C036092:hordothionin protein, Hordeum vulgare; D008239:Lysine