Mutational analysis of the maize gamma zein C-terminal cysteine residues

Alignment of 98 S-rich prolamin amino acid sequences illustrated that four cysteines were preserved nearly 100% and four cysteines were less well conserved. These residues form two to four intramolecular disulfide bonds in several S-rich prolamins. For maize gamma zein, it is unknown whether the eight C-terminal cysteines form intramolecular disulfide bonds. If gamma zein contains intramolecular disulfide bonds, modifications of critical cysteines in soluble thioredoxin -gamma zein fusion protein might prevent disulfide bond formation and result in misfolded and insoluble protein in Escherichia coli. Individual modification of conserved cysteines C128 and C136 resulted in a four-fold reduction in solubility and a significant decrease in expression level compared to wild-type fusion protein. Modification of conserved C156 resulted in a two-fold reduction in expression level but not a significant change in solubility until combined with a non-deleterious Q181R modification. This suggested that C128 and C136 were involved in disulfide bonds critical for protein folding, whereas C156 was more critical for protein stability. Modification of a non-conserved N-terminal cysteine residue (C117) resulted in increased protein solubility, suggesting it was not involved in an intramolecular disulfide bond. From these data and a review of the literature, a disulfide map for gamma zein is proposed. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.