Assembly properties of glycinin subunits: Development of a novel in vitro assembly assay

Craig Duane Dickinson, Purdue University

Abstract

A novel in vitro assembly assay was developed to study the assembly properties of glycinin subunits. The assay used a combination of SP6 transcription and rabbit reticulocyte translation systems to produce subunits in vitro from cDNA. G4 and G5-proglycinin subunits produced by this method self-assembled into trimers which resembled those found in the ER. The system was useful for studying the properties of a precursor-subunit, for testing the effects of modifications, and for studying the functional importance of the subunit family. By testing proglycinin in re-assembly assays with dissociated glycinin, post-translational cleavage was shown to be required for assembly of glycinin precursor-subunits beyond the trimer stage. The results strongly suggest a physiological role for the cleavage. Post-translational processing was postulated to trigger the deposition of 11S seed-storage proteins in vacuolar protein bodies as insoluble aggregates. A series of in-frame deletions were constructed to probe for regions involved in subunit-subunit contacts. Sequences essential for trimer formation mapped to the basic domain of a glycinin subunit while the acidic domain was shown to be important for solubility. A structural model was proposed in which the basic domain is located at the center of a trimer while the acidic sequences are at the outside and exposed to the solvent. 11S storage protein sequences from different plants were aligned and three regions of heterogeneity, which may be good sites for protein engineering, were defined. A series of insertions and deletions were targeted to one such region, the hypervariable region (HVR), and these modifications were tested using various assembly assays. Only minor deleterious effects were observed in the assembly rates of G4-proglycinins with modified HVRs. The results support the suggestion that the region is amenable to modification. Among the alterations tested were insertions which significantly improved the nutritional qualities of a glycinin subunit with regard to sulfur content. Members of the subunit family G2, G4 and G5 were capable of co-assembly into mixed-oligomers, but G2 was not able to self-assemble. Members of the subunit family appear to be functionally interchangeable except that G2 assembly may rely on the presence of other subunits.

Degree

Ph.D.

Advisors

Nielsen, Purdue University.

Subject Area

Biochemistry

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