Structural dynamics of the Streptomyces lividans K+ channel (SKC1): oligomeric stoichiometry and stability.
SKC1, a 160-residue potassium channel with two putative transmembrane (TM) segments was recently identified from Streptomyces lividans. Its high levels of expression, small size, and ease of purification make SKC1 an ideal candidate for high-resolution structural studies. We have initiated the structural characterization of this channel by assessing its oligomeric behavior, stability in detergent, general hydrodynamic properties, and preliminary secondary structure content. SKC1 was readily expressed and purified to homogeneity by sequential metal-chelate and gel filtration chromatography. Standard SDS-PAGE, together with chemical cross-linking analysis indicated that SKC1 behaves as a tightly associated tetramer even in the presence of SDS. Using a gel shift assay to assess its oligomeric state, we determined that SKC1 is stable as a tetramer in most detergents and can be maintained in nonionic detergent solutions for extended periods of time. The tetramer is also stable at relatively high temperatures, with an oligomer-to-monomer transition occurring at approximately 65 degrees C. The Stokes radius of the micellar complex is 5 nm as determined from gel filtration chromatography of SKC1 in dodecyl maltoside. Preliminary estimations of secondary structure from CD spectroscopy showed that the channel exists mostly in alpha-helical conformation, with more than 50% alpha-helical, close to 20% beta-sheet, 10% beta-turn, and about 15% unassigned or random coil. These results are consistent with the idea that a bundle of alpha-helices forming a tetramer around the ion-conductive pathway is the common structural motif for members of the voltage-dependent channel superfamily.
Cortes
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DM
Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, Charlottesville, Virginia 22906-0011, USA.
Perozo
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D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins; D001704:Biopolymers; D003001:Cloning, Molecular; D003902:Detergents; D006358:Hot Temperature; D008969:Molecular Sequence Data; D015221:Potassium Channels / Q000737:chemistry* / Q000235:genetics; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000737:chemistry*; D014981:Xenopus
D001426:Bacterial Proteins; D001704:Biopolymers; D003902:Detergents; D015221:Potassium Channels; C107790:prokaryotic potassium channel