Purification of bacterial membrane sensor kinases and biophysical methods for determination of their ligand and inhibitor interactions
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 met...
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| Format: | Article |
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Portland Press
2016
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| Online Access: | https://eprints.nottingham.ac.uk/41690/ |
| _version_ | 1848796331907219456 |
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| author | 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. |
| author_facet | 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. |
| author_sort | Hussain, Rohanah |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | 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. |
| first_indexed | 2025-11-14T19:46:17Z |
| format | Article |
| id | nottingham-41690 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| last_indexed | 2025-11-14T19:46:17Z |
| publishDate | 2016 |
| publisher | Portland Press |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | nottingham-416902020-05-04T17:57:12Z https://eprints.nottingham.ac.uk/41690/ 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. 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. Portland Press 2016-06-09 Article PeerReviewed Hussain, Rohanah, Harding, Stephen E., Hughes, Charlotte S., Ma, Pikyee, Patching, Simon G., Edara, Shalini, Siligardi, Giuliano, Henderson, Peter J.F. and Phillips-Jones, Mary K. (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 1470-8752 http://www.biochemsoctrans.org/content/44/3/810 doi:10.1042/BST20160023 doi:10.1042/BST20160023 |
| spellingShingle | 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. Purification of bacterial membrane sensor kinases and biophysical methods for determination of their ligand and inhibitor interactions |
| title | Purification of bacterial membrane sensor kinases and biophysical methods for determination of their ligand and inhibitor interactions |
| title_full | Purification of bacterial membrane sensor kinases and biophysical methods for determination of their ligand and inhibitor interactions |
| title_fullStr | Purification of bacterial membrane sensor kinases and biophysical methods for determination of their ligand and inhibitor interactions |
| title_full_unstemmed | Purification of bacterial membrane sensor kinases and biophysical methods for determination of their ligand and inhibitor interactions |
| title_short | Purification of bacterial membrane sensor kinases and biophysical methods for determination of their ligand and inhibitor interactions |
| title_sort | purification of bacterial membrane sensor kinases and biophysical methods for determination of their ligand and inhibitor interactions |
| url | https://eprints.nottingham.ac.uk/41690/ https://eprints.nottingham.ac.uk/41690/ https://eprints.nottingham.ac.uk/41690/ |