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Passenger sequences can promote interlaced dimers in a common variant of the maltose-binding protein
The maltose-binding protein (MBP) is one of the most frequently used protein tags due to its capacity to stabilize, solubilize and even crystallize recombinant proteins that are fused to it. Given that MBP is thought to be a highly stable monomeric protein with known characteristics, fused passenger...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6938514/ https://www.ncbi.nlm.nih.gov/pubmed/31892719 http://dx.doi.org/10.1038/s41598-019-56718-y |
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author | Momin, Afaque A. Hameed, Umar F. Shahul Arold, Stefan T. |
author_facet | Momin, Afaque A. Hameed, Umar F. Shahul Arold, Stefan T. |
author_sort | Momin, Afaque A. |
collection | PubMed |
description | The maltose-binding protein (MBP) is one of the most frequently used protein tags due to its capacity to stabilize, solubilize and even crystallize recombinant proteins that are fused to it. Given that MBP is thought to be a highly stable monomeric protein with known characteristics, fused passenger proteins are often studied without being cleaved from MBP. Here we report that a commonly used engineered MBP version (mutated to lower its surface entropy) can form interlaced dimers when fused to short protein sequences derived from the focal adhesion kinase (FAK) or the homologous protein tyrosine kinase 2 (PYK2). These MBP dimers still bind maltose and can interconvert with monomeric forms in vitro under standard conditions despite a contact surface of more than 11,000 Å(2). We demonstrate that both the mutations in MBP and the fused protein sequences were required for dimer formation. The FAK and PYK2 sequences are less than 40% identical, monomeric, and did not show specific interactions with MBP, suggesting that a variety of sequences can promote this MBP dimerization. MBP dimerization was abrogated by reverting two of the eight mutations introduced in the engineered MBP. Our results provide an extreme example for induced reversible domain-swapping, with implications for protein folding dynamics. Our observations caution that passenger-promoted MBP dimerization might mislead experimental characterization of the fused protein sequences, but also suggest a simple mutation to stop this phenomenon. |
format | Online Article Text |
id | pubmed-6938514 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-69385142020-01-06 Passenger sequences can promote interlaced dimers in a common variant of the maltose-binding protein Momin, Afaque A. Hameed, Umar F. Shahul Arold, Stefan T. Sci Rep Article The maltose-binding protein (MBP) is one of the most frequently used protein tags due to its capacity to stabilize, solubilize and even crystallize recombinant proteins that are fused to it. Given that MBP is thought to be a highly stable monomeric protein with known characteristics, fused passenger proteins are often studied without being cleaved from MBP. Here we report that a commonly used engineered MBP version (mutated to lower its surface entropy) can form interlaced dimers when fused to short protein sequences derived from the focal adhesion kinase (FAK) or the homologous protein tyrosine kinase 2 (PYK2). These MBP dimers still bind maltose and can interconvert with monomeric forms in vitro under standard conditions despite a contact surface of more than 11,000 Å(2). We demonstrate that both the mutations in MBP and the fused protein sequences were required for dimer formation. The FAK and PYK2 sequences are less than 40% identical, monomeric, and did not show specific interactions with MBP, suggesting that a variety of sequences can promote this MBP dimerization. MBP dimerization was abrogated by reverting two of the eight mutations introduced in the engineered MBP. Our results provide an extreme example for induced reversible domain-swapping, with implications for protein folding dynamics. Our observations caution that passenger-promoted MBP dimerization might mislead experimental characterization of the fused protein sequences, but also suggest a simple mutation to stop this phenomenon. Nature Publishing Group UK 2019-12-31 /pmc/articles/PMC6938514/ /pubmed/31892719 http://dx.doi.org/10.1038/s41598-019-56718-y Text en © The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. |
spellingShingle | Article Momin, Afaque A. Hameed, Umar F. Shahul Arold, Stefan T. Passenger sequences can promote interlaced dimers in a common variant of the maltose-binding protein |
title | Passenger sequences can promote interlaced dimers in a common variant of the maltose-binding protein |
title_full | Passenger sequences can promote interlaced dimers in a common variant of the maltose-binding protein |
title_fullStr | Passenger sequences can promote interlaced dimers in a common variant of the maltose-binding protein |
title_full_unstemmed | Passenger sequences can promote interlaced dimers in a common variant of the maltose-binding protein |
title_short | Passenger sequences can promote interlaced dimers in a common variant of the maltose-binding protein |
title_sort | passenger sequences can promote interlaced dimers in a common variant of the maltose-binding protein |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6938514/ https://www.ncbi.nlm.nih.gov/pubmed/31892719 http://dx.doi.org/10.1038/s41598-019-56718-y |
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