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Secondary Structure Prediction of Protein Constructs Using Random Incremental Truncation and Vacuum-Ultraviolet CD Spectroscopy
A novel uracil-DNA degrading protein factor (termed UDE) was identified in Drosophila melanogaster with no significant structural and functional homology to other uracil-DNA binding or processing factors. Determination of the 3D structure of UDE is excepted to provide key information on the descript...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4896422/ https://www.ncbi.nlm.nih.gov/pubmed/27273007 http://dx.doi.org/10.1371/journal.pone.0156238 |
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author | Pukáncsik, Mária Orbán, Ágnes Nagy, Kinga Matsuo, Koichi Gekko, Kunihiko Maurin, Damien Hart, Darren Kézsmárki, István Vertessy, Beata G. |
author_facet | Pukáncsik, Mária Orbán, Ágnes Nagy, Kinga Matsuo, Koichi Gekko, Kunihiko Maurin, Damien Hart, Darren Kézsmárki, István Vertessy, Beata G. |
author_sort | Pukáncsik, Mária |
collection | PubMed |
description | A novel uracil-DNA degrading protein factor (termed UDE) was identified in Drosophila melanogaster with no significant structural and functional homology to other uracil-DNA binding or processing factors. Determination of the 3D structure of UDE is excepted to provide key information on the description of the molecular mechanism of action of UDE catalysis, as well as in general uracil-recognition and nuclease action. Towards this long-term aim, the random library ESPRIT technology was applied to the novel protein UDE to overcome problems in identifying soluble expressing constructs given the absence of precise information on domain content and arrangement. Nine constructs of UDE were chosen to decipher structural and functional relationships. Vacuum ultraviolet circular dichroism (VUVCD) spectroscopy was performed to define the secondary structure content and location within UDE and its truncated variants. The quantitative analysis demonstrated exclusive α-helical content for the full-length protein, which is preserved in the truncated constructs. Arrangement of α-helical bundles within the truncated protein segments suggested new domain boundaries which differ from the conserved motifs determined by sequence-based alignment of UDE homologues. Here we demonstrate that the combination of ESPRIT and VUVCD spectroscopy provides a new structural description of UDE and confirms that the truncated constructs are useful for further detailed functional studies. |
format | Online Article Text |
id | pubmed-4896422 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-48964222016-06-16 Secondary Structure Prediction of Protein Constructs Using Random Incremental Truncation and Vacuum-Ultraviolet CD Spectroscopy Pukáncsik, Mária Orbán, Ágnes Nagy, Kinga Matsuo, Koichi Gekko, Kunihiko Maurin, Damien Hart, Darren Kézsmárki, István Vertessy, Beata G. PLoS One Research Article A novel uracil-DNA degrading protein factor (termed UDE) was identified in Drosophila melanogaster with no significant structural and functional homology to other uracil-DNA binding or processing factors. Determination of the 3D structure of UDE is excepted to provide key information on the description of the molecular mechanism of action of UDE catalysis, as well as in general uracil-recognition and nuclease action. Towards this long-term aim, the random library ESPRIT technology was applied to the novel protein UDE to overcome problems in identifying soluble expressing constructs given the absence of precise information on domain content and arrangement. Nine constructs of UDE were chosen to decipher structural and functional relationships. Vacuum ultraviolet circular dichroism (VUVCD) spectroscopy was performed to define the secondary structure content and location within UDE and its truncated variants. The quantitative analysis demonstrated exclusive α-helical content for the full-length protein, which is preserved in the truncated constructs. Arrangement of α-helical bundles within the truncated protein segments suggested new domain boundaries which differ from the conserved motifs determined by sequence-based alignment of UDE homologues. Here we demonstrate that the combination of ESPRIT and VUVCD spectroscopy provides a new structural description of UDE and confirms that the truncated constructs are useful for further detailed functional studies. Public Library of Science 2016-06-07 /pmc/articles/PMC4896422/ /pubmed/27273007 http://dx.doi.org/10.1371/journal.pone.0156238 Text en © 2016 Pukáncsik et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Pukáncsik, Mária Orbán, Ágnes Nagy, Kinga Matsuo, Koichi Gekko, Kunihiko Maurin, Damien Hart, Darren Kézsmárki, István Vertessy, Beata G. Secondary Structure Prediction of Protein Constructs Using Random Incremental Truncation and Vacuum-Ultraviolet CD Spectroscopy |
title | Secondary Structure Prediction of Protein Constructs Using Random Incremental Truncation and Vacuum-Ultraviolet CD Spectroscopy |
title_full | Secondary Structure Prediction of Protein Constructs Using Random Incremental Truncation and Vacuum-Ultraviolet CD Spectroscopy |
title_fullStr | Secondary Structure Prediction of Protein Constructs Using Random Incremental Truncation and Vacuum-Ultraviolet CD Spectroscopy |
title_full_unstemmed | Secondary Structure Prediction of Protein Constructs Using Random Incremental Truncation and Vacuum-Ultraviolet CD Spectroscopy |
title_short | Secondary Structure Prediction of Protein Constructs Using Random Incremental Truncation and Vacuum-Ultraviolet CD Spectroscopy |
title_sort | secondary structure prediction of protein constructs using random incremental truncation and vacuum-ultraviolet cd spectroscopy |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4896422/ https://www.ncbi.nlm.nih.gov/pubmed/27273007 http://dx.doi.org/10.1371/journal.pone.0156238 |
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