Design, production and characterisation of a thermally-stable mutant of the bacterial voltage gated sodium channel Nachbac
O’Reilly, Andrias O. and Charalambous, Kalypso and Nurani, G. and Wallace, Bonnie A. (2009) Design, production and characterisation of a thermally-stable mutant of the bacterial voltage gated sodium channel Nachbac. Biophysical Journal 96 (3(S1)), 253a-253a. ISSN 0006-3495.
NaChBac from B. halodurans is a bacterial homologue of the eukaryotic voltage-gated sodium channels which has been expressed and purified from E. coli. We have previously shown (Nurani et al (2008) Biochemistry 31:8114-8121) that this membrane protein,, purified from E. coli, forms a mostly helical, tetrameric detergent-solubilisable protein that is capable of binding the drug mibefradil and inducing sodium flux when reconstituted into vesicles. The tetrameric quaternary structure of NaChBac differentiates it from the single-chain eukaryotic sodium channels. The aim of the present study was to produce a more thermally-stable version of this ion channel which would be suitable for a wide range of structural and functional studies. Using molecular modelling techniques, we have designed a mutant, G219S, which incorporates a serine instead of a glycine at the proposed site which is proposed to form the hinge which enables opening and closing of the channel. The aim was to reduce flexibility and “lock” the protein in a single state. Mutant protein was cloned, expressed and purified from E. coli and compared with the wild type protein isolated in the same manner. Whilst it had a similar secondary structure, thermal melting curves monitored by circular dichroism spectroscopy indicated that the mutant was considerably more stable than the wild type protein, although it is still capable of binding mibefradil. Thus the protein produced had the properties as designed and is a particularly suitable candidate for new structural, functional and drug binding studies.
|School:||Birkbeck Schools and Departments > School of Science > Biological Sciences|
|Research Centre:||Bioinformatics, Bloomsbury Centre for, Structural Molecular Biology, Institute of (ISMB)|
|Date Deposited:||04 Aug 2010 14:09|
|Last Modified:||07 Dec 2016 15:05|
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