Plant Science 162 (2002) 131–141 Mutational analysis of the maize gamma zein C-terminal cysteine residues Stephanie C. Ems-McClung 1 , Mustapha Benmoussa, Bryan E. Hainline * Department of Pediatrics, School of Medicine, Indiana Uniersity, Indianapolis, IN, USA Received 17 July 2001; received in revised form 27 September 2001; accepted 28 September 2001 Abstract 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. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Gamma zein; Intramolecular disulfide bonds; Cysteine; Genetic engineering; Phenylketonuria www.elsevier.com/locate/plantsci 1. Introduction Gamma zein, a corn prolamin, was chosen as a candidate for the genetic engineering of a phenylala- nine-free dietary supplement for the treatment of indi- viduals with phenylketonuria (PKU) [1 – 3]. A natural protein genetically engineered to lack phenylalanine might be a suitable dietary supplement that could be baked and heated to produce high quality solid food for the treatment of PKU [1,3]. In order to produce a soluble phenylalanine-free protein, it is necessary to understand the constraints imposed by the sequence of this unusual protein. The grain or caryopsis of cereals, e.g. corn (maize), wheat, rice and oats, contains the plant embryo, the seed coat, and the endosperm. The endosperm con- tains storage proteins that are classified as globulins, prolamins or albumins [4]. The prolamin superfamily includes the -amylase/trypsin inhibitors, the albu- mins, and the prolamins [5]. These proteins contain conserved regions called A, B, and C that have 4 – 8 highly conserved cysteine residues that are be- lieved to have arisen from the triplication of an an- cestral domain followed by insertions and deletions [6,7]. The most divergent class contains the -amy- lase/trypsin inhibitors of maize, barley, wheat, rye, and Indian finger and pearl millet [7]. Indian finger millet and wheat -amylase/trypsin inhibitors contain five intramolecular disulfide bonds between the con- served regions [7,8]. The 2S albumins of dicots are synthesized as a single polypeptide that may be cleaved into two chains and/or linked by disulfide bonds [5,9]. * Corresponding author. Present address: Section of Pediatric Metabolism and Genetics, J.W. Riley Hospital for Children, Rm 0907, 702 Barnhill Drive, Indianapolis, IN 46202-5225, USA. Tel.: +1-317-274-3966; fax: +1-317-278-0936. E-mail address: bhainlin@iupui.edu (B.E. Hainline). 1 Present address: Department of Biochemistry, Medical Sciences, Indiana University, Bloomington, IN, USA. 0168-9452/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII:S0168-9452(01)00549-0