Gallagher, C;
Burlina, F;
Offer, J;
Ramos, A;
(2017)
A method for the unbiased and efficient segmental labelling of RNA-binding proteins for structure and biophysics.
Scientific Reports
, 7
, Article 14083. 10.1038/s41598-017-13950-8.
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Abstract
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.
| Type: | Article |
|---|---|
| Title: | A method for the unbiased and efficient segmental labelling of RNA-binding proteins for structure and biophysics |
| Location: | England |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1038/s41598-017-13950-8 |
| Publisher version: | http://doi.org/10.1038/s41598-017-13950-8 |
| Language: | English |
| Additional information: | Copyright © 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. Te 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/. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Structural and Molecular Biology |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10037575 |
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