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A method for the unbiased and efficient segmental labelling of RNA-binding proteins for structure and biophysics

Most eukaryotic RNA regulators recognise their RNA and protein partners by the combinatorial use of several RNA binding domains. Inter-domain dynamics and interactions play a key role in recognition and can be analysed by techniques such as NMR or FRET, provided that the information relative to the...

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Autores principales: Gallagher, Christopher, Burlina, Fabienne, Offer, John, Ramos, Andres
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5658380/
https://www.ncbi.nlm.nih.gov/pubmed/29074846
http://dx.doi.org/10.1038/s41598-017-13950-8
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author Gallagher, Christopher
Burlina, Fabienne
Offer, John
Ramos, Andres
author_facet Gallagher, Christopher
Burlina, Fabienne
Offer, John
Ramos, Andres
author_sort Gallagher, Christopher
collection PubMed
description Most eukaryotic RNA regulators recognise their RNA and protein partners by the combinatorial use of several RNA binding domains. Inter-domain dynamics and interactions play a key role in recognition and can be analysed by techniques such as NMR or FRET, provided that the information relative to the individual interactions can be de-convoluted. Segmentally labelling the proteins by ligating labelled and unlabelled peptide chains allows one to filter out unwanted information and observe the labelled moieties only. Several strategies have been implemented to ligate two protein fragments, but multiple ligations, which are necessary to segmentally label proteins of more than two domains, are more challenging and often dependent on the structure and solubility of the domains. Here we report a method to ligate multiple protein segments that allows the fast, high yield labelling of both internal and end domains, depending on the requirements. We use TCEP and mercaptophenylacetic acid (MPAA) in an optimised reaction environment to achieve an efficient ligation of protein domains independently from their structure or solubility. We expect the method will provide a useful tool for the molecular study of combinatorial protein–RNA recognition in RNA regulation.
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spelling pubmed-56583802017-10-31 A method for the unbiased and efficient segmental labelling of RNA-binding proteins for structure and biophysics Gallagher, Christopher Burlina, Fabienne Offer, John Ramos, Andres Sci Rep Article Most eukaryotic RNA regulators recognise their RNA and protein partners by the combinatorial use of several RNA binding domains. Inter-domain dynamics and interactions play a key role in recognition and can be analysed by techniques such as NMR or FRET, provided that the information relative to the individual interactions can be de-convoluted. Segmentally labelling the proteins by ligating labelled and unlabelled peptide chains allows one to filter out unwanted information and observe the labelled moieties only. Several strategies have been implemented to ligate two protein fragments, but multiple ligations, which are necessary to segmentally label proteins of more than two domains, are more challenging and often dependent on the structure and solubility of the domains. Here we report a method to ligate multiple protein segments that allows the fast, high yield labelling of both internal and end domains, depending on the requirements. We use TCEP and mercaptophenylacetic acid (MPAA) in an optimised reaction environment to achieve an efficient ligation of protein domains independently from their structure or solubility. We expect the method will provide a useful tool for the molecular study of combinatorial protein–RNA recognition in RNA regulation. Nature Publishing Group UK 2017-10-26 /pmc/articles/PMC5658380/ /pubmed/29074846 http://dx.doi.org/10.1038/s41598-017-13950-8 Text en © The Author(s) 2017 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
Gallagher, Christopher
Burlina, Fabienne
Offer, John
Ramos, Andres
A method for the unbiased and efficient segmental labelling of RNA-binding proteins for structure and biophysics
title A method for the unbiased and efficient segmental labelling of RNA-binding proteins for structure and biophysics
title_full A method for the unbiased and efficient segmental labelling of RNA-binding proteins for structure and biophysics
title_fullStr A method for the unbiased and efficient segmental labelling of RNA-binding proteins for structure and biophysics
title_full_unstemmed A method for the unbiased and efficient segmental labelling of RNA-binding proteins for structure and biophysics
title_short A method for the unbiased and efficient segmental labelling of RNA-binding proteins for structure and biophysics
title_sort method for the unbiased and efficient segmental labelling of rna-binding proteins for structure and biophysics
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5658380/
https://www.ncbi.nlm.nih.gov/pubmed/29074846
http://dx.doi.org/10.1038/s41598-017-13950-8
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