X-ray diffraction analysis and in vitro characterization of the UAM2 protein from Oryza sativa

Ditte Hededam Welner, Alex Yi-Lin Tsai, Andy M. DeGiovanni, Henrik Vibe Scheller, Paul D. Adams

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Abstract

The role of seemingly non-enzymatic proteins in complexes interconverting UDP-arabinopyranose and UDP-arabinofuranose (UDP-arabinosemutases; UAMs) in the plant cytosol remains unknown. To shed light on their function, crystallographic and functional studies of the seemingly non-enzymatic UAM2 protein from Oryza sativa (OsUAM2) were undertaken. Here, X-ray diffraction data are reported, as well as analysis of the oligomeric state in the crystal and in solution. OsUAM2 crystallizes readily but forms highly radiation-sensitive crystals with limited diffraction power, requiring careful low-dose vector data acquisition. Using size-exclusion chromatography, it is shown that the protein is monomeric in solution. Finally, limited proteolysis was employed to demonstrate DTT-enhanced proteolytic digestion, indicating the existence of at least one intramolecular disulfide bridge or, alternatively, a requirement for a structural metal ion.
Original languageEnglish
JournalActa Crystallographica. Section F: Structural Biology and Crystallization Communications
Volume73
Pages (from-to)241-245
ISSN2053-230X
DOIs
Publication statusPublished - 2017

Keywords

  • Reversibly glycosylated polypeptide
  • Limited proteolysis
  • UDP-arabinopyranose mutase
  • Vector data collection

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