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Production of membrane proteins for characterisation of their pheromone-sensing and antimicrobial resistance functions

Azam, Aalishaa A.; Kinder, Jean M.; Khan, G. Nasir; Alase, Ade; Ma, Pikyee; Liu, Yang; Ault, James R.; Henderson, Peter J. F.; Chowdhry, Babur Z.; Alexander, Bruce D.; Harding, Stephen E.; Phillips-Jones, Mary K.

Authors

Aalishaa A. Azam

Jean M. Kinder

G. Nasir Khan

Ade Alase

Pikyee Ma

Yang Liu

James R. Ault

Peter J. F. Henderson

Babur Z. Chowdhry

Bruce D. Alexander

Stephen E. Harding

Dr MARY PHILLIPS-JONES Mary.Phillips-Jones@nottingham.ac.uk
Associate Professor in Polymer & Microbial Biophysics



Abstract

Despite the importance of membrane proteins in cellular processes, studies of these hydrophobic proteins present major technical challenges, including expression and purification for structural and biophysical studies. A modified strategy of that proposed previously by Saidijam et al. (2005) and others, for the routine expression of bacterial membrane proteins involved in environmental sensing and antimicrobial resistance (AMR), is proposed which results in purification of sufficient proteins for biophysical experiments. We report expression successes amongst a collection of enterococcal vancomycin resistance membrane proteins: VanTG, VanTG-M transporter domain, VanZ and the previously characterised VanS (A-type) histidine protein kinase (HPK). Using the same strategy, we report on the successful amplification and purification of intact BlpH and ComD2 HPKs of Streptococcus pneumoniae. Near-UV circular dichroism revealed both recombinant proteins bound their pheromone ligands BlpC and CSP2. Interestingly, CSP1 also interacted with ComD. Finally, we evaluate the alternative strategy for studying sensory HPKs involving isolated soluble sensory domain fragments, exemplified by successful production of VicKESD of Enterococcus faecalis VicK. Purified VicKESD possessed secondary structure post-purification. Thermal denaturation experiments using far-UV CD, a technique which can be revealing regarding ligand binding, revealed that: (a) VicKESD denaturation occurs between 15 and 50 °C; and (b) reducing conditions did not detectably affect denaturation profiles suggesting reducing conditions per se are not directly sensed by VicKESD. Our findings provide information on a modified strategy for the successful expression, production and/or storage of bacterial membrane HPKs, AMR proteins and sensory domains for their future crystallisation, and ligand binding studies.

Journal Article Type Article
Publication Date Jul 31, 2018
Journal European Biophysics Journal
Print ISSN 0175-7571
Electronic ISSN 1432-1017
Publisher Springer Verlag
Peer Reviewed Peer Reviewed
APA6 Citation Azam, A. A., Kinder, J. M., Khan, G. N., Alase, A., Ma, P., Liu, Y., …Phillips-Jones, M. K. (2018). Production of membrane proteins for characterisation of their pheromone-sensing and antimicrobial resistance functions. European Biophysics Journal, doi:10.1007/s00249-018-1325-z
DOI https://doi.org/10.1007/s00249-018-1325-z
Keywords Histidine kinase; Membrane proteins; Vancomycin resistance; Enterococci; Pheromone sensors; Streptococcus pneumoniae; Circular dichroism spectroscopy; Analytical ultracentrifugation
Publisher URL https://link.springer.com/article/10.1007/s00249-018-1325-z

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