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. Mai ze, in particular, is deficient in lysine. The inherent lack of lysine -rich proteins in maize has necessitated the search for heterologous p roteins enriched in this amino acid, the isolation of the correspondin g gene and its ultimate introduction into maize through plant transfor mation techniques. However, a rate-limiting step to this strategy has been the availability of plant-derived lysine-rich proteins. An appeal ing solution to the problem is to artificially increase the lysine con tent of a given protein by mutating appropriate residues to lysine. He re, we expound this strategy, starting with the protein alpha-hordothi onin that is derived from barley seeds and consists of five lysine res idues in a total of 45 amino acids (11% lysine). To facilitate rationa l substitutions, the 3-D structure of the protein has been determined by homology modeling with crambin. Based on this model, we have identi fied surface residues amenable to substitution with lysine. Furthermor e, the acceptability of the mutations has been validated through the s ynthesis and characterization of the derivatives. To this end, our app roach has permitted the creation of a modified alpha-hordothionin prot ein that has a lysine content of similar to 27% and retains the antifu ngal activity of the wild-type protein.