Role of individual disulfide bonds in the structural maturation of a low molecular weight glutenin subunit

Gliadins and glutenins are the major storage proteins that accumulate in wheat
endosperm cells during seed development. Although gliadins are mainly monomeric,
glutenins consist of very large disulfide-linked polymers made up of high
molecular weight and low molecular weight subunits. These polymers are among the
largest protein molecules known in nature and are the most important
determinants of the viscoelastic properties of gluten. As a first step toward
the elucidation of the folding and assembly pathways that lead to glutenin
polymer formation, we have exploited an in vitro system composed of wheat germ
extract and bean microsomes to examine the role of disulfide bonds in the
structural maturation of a low molecular weight glutenin subunit. When
conditions allowing the formation of disulfide bonds were established, the in
vitro synthesized low molecular weight glutenin subunit was recovered in
monomeric form containing intrachain disulfide bonds. Conversely, synthesis
under conditions that did not favor the formation of disulfide bonds led to the
production of large aggregates from which the polypeptides could not be rescued
by the post-translational generation of a more oxidizing environment. These
results indicate that disulfide bond formation is essential for the
conformational maturation of the low molecular weight glutenin subunit and
suggest that early folding steps may play an important role in this process,
allowing the timely pairing of critical cysteine residues. To determine which
cysteines were important to maintain the protein in monomeric form, we prepared
a set of mutants containing selected cysteine to serine substitutions. Our
results show that two conserved cysteine residues form a critical disulfide bond
that is essential in preventing the exposure of adhesive domains and the
consequent formation of aberrant aggregates.