Expression and characterization of the N-terminal domain of an oleosin protein from sunflower

Oil bodies of plant seeds contain a triacylglycerol matrix surrounded by a monolayer of phospholipids embedded with alkaline proteins termed oleosins. Although oleosins are amphipathic proteins, they are unlike bilayer membrane proteins since they are associated with a single lipid:water interface at the oil body surface. Oleosins are unusual proteins because they contain a 70-80-residue uninterrupted nonpolar domain, flanked by relative polar C- and N-terminal domains. In the present study, we report the expression of the N-terminal domain of the 18-kDa oleosin isoform from sunflower as a recombinant fusion protein in Escherichia coli and the determination of its secondary structure using CD and Fourier transform infrared spectroscopy either as a purified but partially denatured peptide or reconstituted into liposomes. The structure derived from physical studies was then compared and assigned with those predicted from analysis of the primary sequence of the N-terminal domain. Based on data derived from CD spectroscopy analysis of purified and partially renatured N-terminal polypeptide, it contains about 10% alpha-helical structure, 20-30% beta-strand structure, approximately 8% beta-turn structure, and 60% random coil structure. However, analysis of the polypeptide reconstituted into liposomes showed an increased content of alpha-helical structure to about 20% and an increased beta-strand structure content to about 30-40%. Data derived from Fourier transform infrared spectroscopy studies and compared with the data predicted from the primary sequence showed the peptide is well structured with some antiparallel beta-strand structure from residues 2-9, parallel beta-strand structure from residues 30-37 and/or 42-49, and alpha-helical structure from residues 10-23 and/or 43-49. There is potential amphipathic alpha-helix from residues 10-23. Based on these results, the following model for the secondary structure of the N-terminal domain of sunflower oleosin can be proposed. Residues 2-9 would produce amphipathic antiparallel beta-strand structure. Residues 10-23 would produce an amphipathic alpha-helical structure. Residues 30-37 and/or 42-49 would give parallel beta-strand structure, or residues 42-49 could form a nonpolar alpha-helical structure that would insert into the oil matrix.