Morrison, PM, Balmforth, MR, Ness, SW et al. (4 more authors) (2016) Confirmation of a protein-protein interaction in the pantothenate biosynthetic pathway using Sortase-mediated labelling. ChemBioChem, 17 (8). pp. 753-758. ISSN 1439-4227
Abstract
High-throughput studies have been widely used to identify protein-protein interactions however the veracity of few of these candidate interactions have been demonstrated in vitro. We use a combination of isothermal titration calorimetry and fluorescence anisotropy to screen candidate interactions within the pantothenate biosynthetic pathway. In particular, we observe no interaction between the subsequent enzyme in the pathway, pantothenate synthetase (PS) and aspartate decarboxylase but do observe interaction of PS and the putative Nudix hydrolase, YfcD. Confirmation of the interaction by fluorescence anisotropy was dependent upon labelling of an adventitiously formed glycine on the protein N-terminal affinity purification tag using Sortase. Subsequent formation of the protein-protein complex led to apparent restriction of the dynamics of this tag, suggesting that this approach could be generally applied to a subset of other protein-protein interaction complexes.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. This is the peer-reviewed version of the following article: P. M. Morrison, M. R. Balmforth, S. W. Ness, D. J. Williamson, M. D. Rugen, W. B. Turnbull, M. E. Webb, ChemBioChem 2016, 17, 753, which has been published in final form at http://dx.doi.org/10.1002/cbic.201500547. This article may be used for non-commercial purposes in accordance with Wiley-VCH Terms and Conditions for Self-Archiving. |
Keywords: | fluorescence anisotropy; pantothenate biosynthesis; sortase labeling |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Biological Sciences (Leeds) The University of Leeds > Faculty of Engineering & Physical Sciences (Leeds) > School of Chemistry (Leeds) > Organic Chemistry (Leeds) |
Depositing User: | Symplectic Publications |
Date Deposited: | 28 Jan 2016 16:27 |
Last Modified: | 14 Apr 2017 03:05 |
Published Version: | http://dx.doi.org/10.1002/cbic.201500547 |
Status: | Published |
Publisher: | Wiley-VCH Verlag |
Identification Number: | 10.1002/cbic.201500547 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:94327 |