Unfolding of a ClC chloride transporter retains memory of its evolutionary history

ClC chloride channels and transporters are important for chloride homeostasis in species from bacteria to human. Mutations in ClC proteins cause genetically inherited diseases, some of which are likely to have folding defects. The ClC proteins present a challenging and unusual biological folding problem because they are large membrane proteins possessing a complex architecture with many re-entrant helices that go only part way through membrane and loop back out. Here we were able to examine the unfolding of the E. coli ClC transporter, ClC-ec1, using single-molecule forced unfolding methods. We find that the protein can be separated into two stable halves that unfold independently. The independence of the two domains is consistent with an evolutionary model in which the two halves arose from independent folding subunits that later fused together. Maintaining smaller folding domains of lesser complexity within large membrane proteins may be an advantageous strategy to avoid misfolding traps.