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

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.

Authors:
ORCiD logo; ; ; ORCiD logo;
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
OSTI Identifier:
1349149
Alternate Identifier(s):
OSTI ID: 1393137
Grant/Contract Number:  
AC02-05CH11231
Resource Type:
Published Article
Journal Name:
Acta Crystallographica. Section F, Structural Biology Communications
Additional Journal Information:
Journal Name: Acta Crystallographica. Section F, Structural Biology Communications Journal Volume: 73 Journal Issue: 4; Journal ID: ISSN 2053-230X
Publisher:
International Union of Crystallography (IUCr)
Country of Publication:
United Kingdom
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; reversibly glycosylated polypeptide; limited proteolysis; UDP-arabinopyranose mutase; vector data collection

Citation Formats

Welner, Ditte Hededam, Tsai, Alex Yi-Lin, DeGiovanni, Andy M., Scheller, Henrik Vibe, and Adams, Paul D. X-ray diffraction analysis and in vitro characterization of the UAM2 protein from Oryza sativa. United Kingdom: N. p., 2017. Web. doi:10.1107/S2053230X17004587.
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Welner, Ditte Hededam, Tsai, Alex Yi-Lin, DeGiovanni, Andy M., Scheller, Henrik Vibe, & Adams, Paul D. X-ray diffraction analysis and in vitro characterization of the UAM2 protein from Oryza sativa. United Kingdom. https://doi.org/10.1107/S2053230X17004587
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Welner, Ditte Hededam, Tsai, Alex Yi-Lin, DeGiovanni, Andy M., Scheller, Henrik Vibe, and Adams, Paul D. Wed . "X-ray diffraction analysis and in vitro characterization of the UAM2 protein from Oryza sativa". United Kingdom. https://doi.org/10.1107/S2053230X17004587.
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@article{osti_1349149,
title = {X-ray diffraction analysis and in vitro characterization of the UAM2 protein from Oryza sativa},
author = {Welner, Ditte Hededam and Tsai, Alex Yi-Lin and DeGiovanni, Andy M. and Scheller, Henrik Vibe and Adams, Paul D.},
abstractNote = {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.},
doi = {10.1107/S2053230X17004587},
journal = {Acta Crystallographica. Section F, Structural Biology Communications},
number = 4,
volume = 73,
place = {United Kingdom},
year = {2017},
month = {3}
}
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Journal Article:
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https://doi.org/10.1107/S2053230X17004587
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