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Bacillus subtilis MraY in detergent-free system of nanodiscs wrapped by styrene-maleic acid copolymers

As an integral membrane protein, purification and characterization of phospho-N- acetylmuramyl- pentapeptide translocase MraY have proven difficult. Low yield and concerns of retaining stability and activity after detergent solubilization have hampered the structure-function analysis. The recently d...

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Autores principales: Liu, Yao, Moura, Elisabete C. C. M., Dörr, Jonas M., Scheidelaar, Stefan, Heger, Michal, Egmond, Maarten R., Killian, J. Antoinette, Mohammadi, Tamimount, Breukink, Eefjan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6218056/
https://www.ncbi.nlm.nih.gov/pubmed/30395652
http://dx.doi.org/10.1371/journal.pone.0206692
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author Liu, Yao
Moura, Elisabete C. C. M.
Dörr, Jonas M.
Scheidelaar, Stefan
Heger, Michal
Egmond, Maarten R.
Killian, J. Antoinette
Mohammadi, Tamimount
Breukink, Eefjan
author_facet Liu, Yao
Moura, Elisabete C. C. M.
Dörr, Jonas M.
Scheidelaar, Stefan
Heger, Michal
Egmond, Maarten R.
Killian, J. Antoinette
Mohammadi, Tamimount
Breukink, Eefjan
author_sort Liu, Yao
collection PubMed
description As an integral membrane protein, purification and characterization of phospho-N- acetylmuramyl- pentapeptide translocase MraY have proven difficult. Low yield and concerns of retaining stability and activity after detergent solubilization have hampered the structure-function analysis. The recently developed detergent-free styrene-maleic acid (SMA) co-polymer system offers an alternative approach that may overcome these disadvantages. In this study, we used the detergent free system to purify MraY from Bacillus subtilis. This allowed efficient extraction of MraY that was heterologously produced in Escherichia coli membranes into SMA-wrapped nanodiscs. The purified MraY embedded in these nanodiscs (SMA-MraY) was comparable to the micellar MraY extracted with a conventional detergent (DDM) with regard to the yield and the purity of the recombinant protein but required significantly less time. The predominantly alpha-helical secondary structure of the protein in SMA-wrapped nanodiscs was also more stable against heat denaturation compared to the micellar protein. Thus, this detergent-free system is amenable to extract MraY efficiently and effectively while maintaining the biophysical properties of the protein. However, the apparent activity of the SMA-MraY was reduced compared to that of the detergent-solubilized protein. The present data indicates that this is caused by a lower accessibility of the enzyme in SMA-wrapped nanodiscs towards its polyisoprenoid substrate.
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spelling pubmed-62180562018-11-19 Bacillus subtilis MraY in detergent-free system of nanodiscs wrapped by styrene-maleic acid copolymers Liu, Yao Moura, Elisabete C. C. M. Dörr, Jonas M. Scheidelaar, Stefan Heger, Michal Egmond, Maarten R. Killian, J. Antoinette Mohammadi, Tamimount Breukink, Eefjan PLoS One Research Article As an integral membrane protein, purification and characterization of phospho-N- acetylmuramyl- pentapeptide translocase MraY have proven difficult. Low yield and concerns of retaining stability and activity after detergent solubilization have hampered the structure-function analysis. The recently developed detergent-free styrene-maleic acid (SMA) co-polymer system offers an alternative approach that may overcome these disadvantages. In this study, we used the detergent free system to purify MraY from Bacillus subtilis. This allowed efficient extraction of MraY that was heterologously produced in Escherichia coli membranes into SMA-wrapped nanodiscs. The purified MraY embedded in these nanodiscs (SMA-MraY) was comparable to the micellar MraY extracted with a conventional detergent (DDM) with regard to the yield and the purity of the recombinant protein but required significantly less time. The predominantly alpha-helical secondary structure of the protein in SMA-wrapped nanodiscs was also more stable against heat denaturation compared to the micellar protein. Thus, this detergent-free system is amenable to extract MraY efficiently and effectively while maintaining the biophysical properties of the protein. However, the apparent activity of the SMA-MraY was reduced compared to that of the detergent-solubilized protein. The present data indicates that this is caused by a lower accessibility of the enzyme in SMA-wrapped nanodiscs towards its polyisoprenoid substrate. Public Library of Science 2018-11-05 /pmc/articles/PMC6218056/ /pubmed/30395652 http://dx.doi.org/10.1371/journal.pone.0206692 Text en © 2018 Liu 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
Liu, Yao
Moura, Elisabete C. C. M.
Dörr, Jonas M.
Scheidelaar, Stefan
Heger, Michal
Egmond, Maarten R.
Killian, J. Antoinette
Mohammadi, Tamimount
Breukink, Eefjan
Bacillus subtilis MraY in detergent-free system of nanodiscs wrapped by styrene-maleic acid copolymers
title Bacillus subtilis MraY in detergent-free system of nanodiscs wrapped by styrene-maleic acid copolymers
title_full Bacillus subtilis MraY in detergent-free system of nanodiscs wrapped by styrene-maleic acid copolymers
title_fullStr Bacillus subtilis MraY in detergent-free system of nanodiscs wrapped by styrene-maleic acid copolymers
title_full_unstemmed Bacillus subtilis MraY in detergent-free system of nanodiscs wrapped by styrene-maleic acid copolymers
title_short Bacillus subtilis MraY in detergent-free system of nanodiscs wrapped by styrene-maleic acid copolymers
title_sort bacillus subtilis mray in detergent-free system of nanodiscs wrapped by styrene-maleic acid copolymers
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6218056/
https://www.ncbi.nlm.nih.gov/pubmed/30395652
http://dx.doi.org/10.1371/journal.pone.0206692
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