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Purification of bacterial membrane sensor kinases and biophysical methods for determination of their ligand and inhibitor interactions

Hussain, Rohanah; Harding, Stephen E.; Hughes, Charlotte S.; Ma, Pikyee; Patching, Simon G.; Edara, Shalini; Siligardi, Giuliano; Henderson, Peter J.F.; Phillips-Jones, Mary K.

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Authors

Rohanah Hussain

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STEPHEN HARDING STEVE.HARDING@NOTTINGHAM.AC.UK
Professor of Applied Biochemistry

Charlotte S. Hughes

Pikyee Ma

Simon G. Patching

Shalini Edara

Giuliano Siligardi

Peter J.F. Henderson

Mary K. Phillips-Jones



Abstract

This article reviews current methods for the reliable heterologous overexpression in Escherichia coli and purification of milligram quantities of bacterial membrane sensor kinase (MSK) 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 MSKs and other membrane proteins–synchrotron radiation circular dichroism (SRCD) spectroscopy and analytical ultracentrifugation (AUC), both of which are non-immobilization and matrix-free methods that require no labelling strategies. Other techniques such as isothermal titration calorimetry (ITC) 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, oligomerization 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 characterize inhibitor binding and any associated protein conformational changes, thereby contributing valuable information for future drug intervention strategies directed towards bacterial MSKs.

Citation

Hussain, R., Harding, S. E., Hughes, C. S., Ma, P., Patching, S. G., Edara, S., …Phillips-Jones, M. K. (2016). Purification of bacterial membrane sensor kinases and biophysical methods for determination of their ligand and inhibitor interactions. Biochemical Society Transactions, 44(3), 810-823. https://doi.org/10.1042/BST20160023

Journal Article Type Article
Acceptance Date Jan 15, 2016
Online Publication Date Jun 9, 2016
Publication Date Jun 15, 2016
Deposit Date Apr 3, 2017
Publicly Available Date Apr 3, 2017
Journal Biochemical Society Transactions
Print ISSN 0300-5127
Electronic ISSN 1470-8752
Publisher Portland Press
Peer Reviewed Peer Reviewed
Volume 44
Issue 3
Pages 810-823
DOI https://doi.org/10.1042/BST20160023
Public URL https://nottingham-repository.worktribe.com/output/795985
Publisher URL http://www.biochemsoctrans.org/content/44/3/810

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