Purification and structural characterization of the central hydrophobic domain of oleosin

Ming Li*, Denis J. Murphy, Ka Ho K. Lee, Reginald Wilson, Linda J. Smith, David C. Clark, Jao Yiu Sung

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

55 Citations (Scopus)


The oil bodies of rapeseeds contain a triacylglycerol matrix surrounded by a monolayer of phospholipids embedded with abundant structural alkaline proteins termed oleosins and some other minor proteins. Oleosins are unusual proteins because they contain a 70-80-residue uninterrupted nonpolar domain flanked by relatively polar C- and N-terminal domains. Although the hydrophilic N-terminal domain had been studied, the structural feature of the central hydrophobic domain remains unclear due to its high hydrophobicity. In the present study, we reported the generation, purification, and characterization of a 9-kDa central hydrophobic domain from rapeseed oleosin (19 kDa). The 9-kDa central hydrophobic domain was produced by selectively degrading the N and C termini with enzymes and then purifying the digest by SDS-PAGE and electroelution. We have also reconstituted the central domain into liposomes and synthetic oil bodies to determine the secondary structure of the domain using CD and Fourier transform infrared (FTIR) spectroscopy. The spectra obtained from CD and FTIR were analyzed with reference to structural information of the N-terminal domain and the full-length rapeseed oleosin. Both CD and FTIR analysis revealed that 50-63% of the domain was composed of β-sheet structure. Detailed analysis of the FTIR spectra indicated that 80% of the β-sheet structure, present in the central domain, was arranged in parallel to the intermolecular β-sheet structure. Therefore, interactions between adjacent oleosin proteins would give rise to a stable β-sheet structure that would extend around the surface of the seed oil bodies stabilizing them in emulsion systems. The strategies used in our present study are significant in that it could be generally used to study difficult proteins with different independent structural domains, especially with long hydrophobic domains.

Original languageEnglish
Pages (from-to)37888-37895
Number of pages8
JournalJournal of Biological Chemistry
Issue number40
Publication statusPublished - 4 Oct 2002
Externally publishedYes


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