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...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2018-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC6218056?pdf=render |
| _version_ | 1817990215779745792 |
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| author | Yao Liu Elisabete C C M Moura Jonas M Dörr Stefan Scheidelaar Michal Heger Maarten R Egmond J Antoinette Killian Tamimount Mohammadi Eefjan Breukink |
| author_facet | Yao Liu Elisabete C C M Moura Jonas M Dörr Stefan Scheidelaar Michal Heger Maarten R Egmond J Antoinette Killian Tamimount Mohammadi Eefjan Breukink |
| author_sort | Yao Liu |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-04-14T00:56:23Z |
| format | Article |
| id | doaj.art-8c3f1fcc5d7a4a379f1551a30d646bc7 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-14T00:56:23Z |
| publishDate | 2018-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-8c3f1fcc5d7a4a379f1551a30d646bc72022-12-22T02:21:36ZengPublic Library of Science (PLoS)PLoS ONE1932-62032018-01-011311e020669210.1371/journal.pone.0206692Bacillus subtilis MraY in detergent-free system of nanodiscs wrapped by styrene-maleic acid copolymers.Yao LiuElisabete C C M MouraJonas M DörrStefan ScheidelaarMichal HegerMaarten R EgmondJ Antoinette KillianTamimount MohammadiEefjan BreukinkAs 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.http://europepmc.org/articles/PMC6218056?pdf=render |
| spellingShingle | Yao Liu Elisabete C C M Moura Jonas M Dörr Stefan Scheidelaar Michal Heger Maarten R Egmond J Antoinette Killian Tamimount Mohammadi Eefjan Breukink Bacillus subtilis MraY in detergent-free system of nanodiscs wrapped by styrene-maleic acid copolymers. PLoS ONE |
| 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 |
| url | http://europepmc.org/articles/PMC6218056?pdf=render |
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