Expression, purification and biophysical characterization of a superfamily of prokaryotic voltage-gated sodium channels
McCusker, Emily C. and Wallace, Bonnie A. (2009) Expression, purification and biophysical characterization of a superfamily of prokaryotic voltage-gated sodium channels. Biophysical Journal 96 (3(S1)), 13a-13a. ISSN 0006-3495.
Eukaryotic voltage-gated sodium channels are monomeric membrane proteins comprised of four pseudo-repeats of domains containing six transmembrane segments, and have a molecular weight of >200 KDa. Their size and complexity makes them an arduous target for production in heterologous expression systems, a necessary step in acquiring the amounts of protein needed for biophysical and structural characterization. The simplified, single domain bacterial sodium channel containing six transmembrane segments isolated from Bacillus halodurans, NaChBac, can be expressed in E. coli in yields suitable for biophysical characterization and may enable successful crystallization and 3-D structure determination. Upon purification in 0.1% DDM, NaChBac is functional and associates to form a stable homotetramer (Nurani et al. (2008) Biochemistry 31:8114-8121). Seven different bacterial sodium channels with significant homology to NaChBac have been expressed in E. coli, purified in high-yield and characterized for secondary structure, thermal stability and drug binding. The experimental ease of obtaining a pure and homogeneous sample varies amongst the superfamily of sodium channels studied. The bacterial sodium channels are extremely thermal stable but individual members differ in their long-term stability when stored at room temperature, 4°C and −80°C, and their ability to bind the drug mibefradil. These channels also differ in their ability to form stable tetramers upon purification in different detergents. The differences and similarities found in this superfamily of sodium channels may prove valuable for determining general structural features important for specific voltage-gated sodium channel functions. The ability to express, purify and reconstitute multiple active bacterial sodium channels in membrane-mimic environments provides an arsenal of resources for elucidating structural features and identifying residues important for sustaining function in voltage-gated sodium channels. (Supported by a grants from the BBSRC to the MPSI consortium and BAW)
|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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