EFFECTS OF DELETION OF DISULFIDE BONDS BY PROTEIN ENGINEERING ON THE CONFORMATION AND FUNCTIONAL-PROPERTIES OF SOYBEAN PROGLYCININ

Glycinin, one of the dominant storage proteins of soybean seeds, has t wo disulfide bonds in each constituent subunit: Cys12-Cys45 and Cys88- Cys298 in the proglycinin A1aB1b subunit. To examine the effects of di srupting disulfide bonds on the formation and maintenance of structure and on the functional properties of proglycinin, we replaced the cyst eine residues (Cysl2 and Cys88) by oligonucleotide-directed mutagenesi s, giving mutant proglycinins Gly12,Ser88, and Gly12Ser88. The mutant proglycinins overproduced in Escherichia coli cells accumulated as sol uble proteins and self-assembled into trimers like the native proglyci nin. The functional properties of proglycinins Gly12 and Ser88 purifie d to near homogeneity were examined as models of modified glycinins. P roglycinin Ser88 formed a harder gel than native glycinin and unmodifi ed expressed proglycinin even at the protein concentrations at which n ative glycinin did not form a hard gel. On the other hand, proglycinin Gly12 formed a gel only at higher protein concentrations (>6%), the h ardness of which was similar to that of the native glycinin. Both prog lycinins Gly12 and Ser88 exhibited emulsifying activity similar to tha t of unmodified expressed proglycinin. These results suggest that the number and topology of free sulfhydryl residues are closely related to the heat-induced gel-forming ability and the gel properties of glycin in but not to its emulsification.