Hussain, R, Harding, SE, Hughes, CS et al. (6 more authors) (2016) Purification of bacterial membrane sensor kinases and biophysical methods for determination of their ligand and inhibitor interactions. Biochemical Society Transactions, 44 (3). pp. 810-823. ISSN 0300-5127
Abstract
This article reviews current methods for the reliable heterologous overexpression in E. coli and purification of milligram quantities of bacterial membrane sensor kinase proteins belonging to the two-component signal transduction family of integral membrane proteins. Many of these methods were developed at Leeds alongside Professor Steve Baldwin to whom this review is dedicated. It also reviews two biophysical methods that we have adapted successfully for studies of purified membrane sensor kinases and other membrane proteins - synchrotron radiation circular dichroism spectroscopy and analytical ultracentrifugation, both of which are non-immobilisation and matrix-free methods that require no labelling strategies. Other techniques such as isothermal titration calorimetry also share these features but generally require high concentrations of material. In common with many other biophysical techniques, both of these biophysical methods provide information regarding membrane protein conformation, oligomerisation state and ligand binding, but they possess the additional advantage of providing direct assessments of whether ligand binding interactions are accompanied by conformational changes. Therefore, both methods provide a powerful means by which to identify and characterise inhibitor binding and any associated protein conformational changes, thereby contributing valuable information for future drug intervention strategies directed towards bacterial membrane sensor kinases.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | (c) 2016 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution Licence 4.0 (CC BY-NC-ND). |
Keywords: | membrane sensor kinase; histidine kinase; two-component system; analytical ultracentrifugation; circular dichroism |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Biomedical Sciences (Leeds) |
Funding Information: | Funder Grant number EU - European Union 201924 |
Depositing User: | Symplectic Publications |
Date Deposited: | 23 Sep 2016 15:29 |
Last Modified: | 04 Nov 2016 07:35 |
Published Version: | http://dx.doi.org/%2010.1042/BST20160023 |
Status: | Published |
Publisher: | Portland Press |
Identification Number: | 10.1042/BST20160023 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:96928 |