STRATEGIES FOR SELECTING MUTATION SITES FOR METHIONINE ENHANCEMENT INTHE BEAN SEED STORAGE PROTEIN PHASEOLIN

The complete three-dimensional structure of the bean seed storage prot ein phaseolin was generated from alpha-carbon coordinates by using mol ecular mechanic calculations. This structure was used as a template to simulate modifications aimed at increasing the methionine content of phaseolin. A hydrophilic, methionine-rich looping insert sequence was designed. Simulated mutagenesis shows that the insert might be accommo dated in turn and loop regions of the protein, but not within an alpha -helix. Methionine content was also increased by the replacement of hy drophobic amino acids with methionine in the central core beta-barrels of the phaseolin protein. Calculations indicated that methionine can effectively replace conserved or variant leucine, isoleucine, and vali ne residues. However, alanine residues were much more sensitive to sub stitution, and demonstrated high variability in the effects of methion ine replacement. Introduction of multiple substitutions in the barrel interior demonstrated that the replaced residues could interact favora bly to relieve local perturbations caused by individual substitutions. Molecular dynamics simulations were also utilized to study the struct ural organization of phaseolin. The calculations indicate that there a re extensive packing interactions between the major domains of phaseol in, which have important implications for protein folding and stabilit y. Since the proposed mutant proteins can be produced and studied, the results presented here provide an ideal test to determine if there is a correlation between the effects obtained by computer simulation and the effects of the mutations on the protein structure expressed in vi vo.