Abstract
The three main isoforms of the 19-kDa lipid body proteins (oleosin) have been purified to homogeneity from embryos of rapeseed. The secondary structures of these proteins as derived from circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy were compared with the secondary structures predicted from the primary sequences. The salient feature of the primary sequence of all oleosins is its division into three defined structural domains: a central hydrophobic domain flanked on either side by relatively hydrophilic domains, respectively. Using a variety of predictive methods based on primary amino acid sequence data, the oleosins exhibited a high probability of beta-strand structure in the 70-residue central hydrophobic domain, with relatively little alpha-helical content. Secondary structure data derived from CD and FTIR were consistent with the predictions from primary sequence, showing that the oleosins contained about 45% beta-strand and 13% alpha-helical structure. Under high salt conditions, a 40-kDa polypeptide was obtained from purified preparations of the 19-kDa oleosins. The 40-kDa polypeptide has a very similar secondary structure, as analyzed by CD and FTIR, to that of the 19-kDa oleosins. This polypeptide is therefore probably a dimer of the 19-kDa oleosins that is formed in high salt environments. A model of the general structure of oleosins is proposed whereby the central hydrophobic domain of the protein with a predominantly beta-strand structure is embedded into the non-aqueous phase of lipid-bodies. This hydrophobic region is flanked by putative alpha-helical structures in the polar N- and C-terminal domains which are probably oriented at the lipid-water interface.
Highlights
The three main isoforms of the 19-kDa lipid body species of oilseeds [2, 3] from widely separated families
The secondary structures of these proteins as derived from circular dichroism (CD) and Fourier transforminfrared (FTIR) spectroscopy were compared withthesecondary structures predicted from the primarysequences
Using a variety of predictive methods based on primary amino acid sequence data, theoleosins exhibited a high probability of &strand structurein the 70-residue central hydrophobic domain, withrelativelylittlea-helical storage in plants, and predictions from their primary amino acid sequencessuggestthat they possess some of the structural features of the transport apolipoproteins, which stabilize the morphologically similar lipid-protein droplets found in the circulatory systems of animals [4]
Summary
Washed three times in 5 volumes of buffer (Tris-HC1,20 mM, pH 9.0). The resulting membrane proteins were recovered by centrifugation at 100,000 X g for 1h. Oil bodymembrane oleosins solubilisedby 1.5% SDS were applied to a column (90 X 2.2 cm) of Sephadex G-75 equilibrated with 30 mM Tris-HC1 buffer, containing 0.2% SDS, pH 9.5, and eluted with the same buffer. Fractions of 2 ml were collected at a flow rate of 20 ml/ h. Fractions containing partly purified 19-kDaoleosins were collected and dialysedagainst 20 mM Tris-HC1,pH 9.5, for 48 h.After extensive dialysis, the proteins were further separated by FPLC on a Mono Q column equilibrated with 30 mM Tris-HC1, pH 9.5. After extensive washing with the equilibration buffer until the protein level was nearly zero, the proteins absorbed onto the column were eluted successively with a linear gradient of 0-1 M NaCl in the equilibration buffer. The purity of oleosins were checked by SDS-PAGE (12%) according to Schagger and Von Jagow [8].The proteins were visualizedwith CoomassieBlue R-250staining. The purified 19-kDa upper, middle, and lower fractions, as identified by their mobility in SDS-PAGE, were used in the following experiments
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