G219S mutagenesis as a means of stabilizing conformational flexibility in the bacterial sodium channel NaChBac.
The NaChBac sodium channel from Bacillus halodurans is a homologue of eukaryotic voltage-gated sodium channels. It can be solubilized in a range of detergents and consists of four identical subunits assembled as a tetramer. Sodium channels are relatively flexible molecules, adopting different conformations in their closed, open and inactivated states. This study aimed to design and construct a mutant version of the NaChBac protein that would insert into membranes and retain its folded conformation, but which would have enhanced stability when subjected to thermal stress. Modelling studies suggested a G219S mutant would have decreased conformational flexibility due to the removal of the glycine hinge around the proposed gating region, thereby imparting increased resistance to unfolding. The mutant expressed in Escherichia coli and purified in the detergent dodecyl maltoside was compared to wildtype NaChBac prepared in a similar manner. The mutant was incorporated into the membrane fraction and had a nearly identical secondary structure to the wildtype protein. When the thermal unfolding of the G219S mutant was examined by circular dichroism spectroscopy, it was shown to not only have a Tm approximately 10 degrees C higher than the wildtype, but also in its unfolded state it retained more ordered helical structure than did the wildtype protein. Hence the G219S mutant was shown to be, as designed, more thermally stable.
O'Reilly
Andrias O
AO
Department of Crystallography, Birkbeck College, University of London, London, UK.
Charalambous
Kalypso
K
Nurani
Ghasem
G
Powl
Andrew M
AM
Wallace
B A
BA
D001407:Bacillus / Q000737:chemistry / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D004926:Escherichia coli / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000096:biosynthesis / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D055550:Protein Stability; D016415:Sequence Alignment; D015222:Sodium Channels / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics
D001426:Bacterial Proteins; D050505:Mutant Proteins; C443666:NaChBac protein, bacteria; D015222:Sodium Channels