EXTENSIVE MODIFICATIONS FOR METHIONINE ENHANCEMENT IN THE BETA-BARRELS DO NOT ALTER THE STRUCTURAL STABILITY OF THE BEAN SEED STORAGE PROTEIN PHASEOLIN

Common beans are widely utilized as a food source, yet are low in the essential amino acid methionine. As an initial step to overcome this d efect the methionine content of the primary bean seed storage protein phaseolin was increased by replacing 20 evolutionarily variant hydroph obic residues with methionine and inserting short, methionine-rich seq uences into turn and loop regions of the protein structure. Methionine enhancement ranged from 5 to 30 residues. An Escherichia coli express ion system was developed to characterize the structural stability of t he mutant proteins. Proteins of expected sizes were obtained for all c onstructs except for negative controls, which were rapidly degraded in E. coli. Thermal denaturation of the purified proteins demonstrated t hat both wild-type and mutant phaseolin proteins denatured reversibly at approximately 61 degrees C. In addition, urea denaturation experime nts of the wild-type and a mutant protein (with 30 additional methioni nes) confirmed that the structural stability of the proteins was very similar. Remarkably, these results indicate that the phaseolin protein tolerates extensive modifications, including 20 substitutions and two loop inserts for methionine enhancement in the P-barrel and loop stru ctures, with extremely small effects on protein stability.