Chloride Intracellular Channels (CLIC) are a family of highly conserved proteins which have been shown to have metamorphic properties. CLICs are able to reversibly auto-insert from the cytosol into the lipid bilayer where they can form active ion channels. A recent next generation resequencing of the X-chromosome revealed a missense mutation (H101Q) in the CLIC2 gene in two related male patients with a known X-Linked Intellectual Disability (XLID). In addition, four non-synonymous single nucleotide polymorphisms (nsSNPs) (S109C, P160A, D161H/Y) were also identified in the CLIC2 gene of healthy individuals. An in silico modelling study examining the effect of these mutations on the structural stability, flexibility and membrane binding properties of the CLIC2 protein was presented in 2011⁽¹⁾. The in silico data suggested that the disease linked mutation, H101Q, located in the hinge region between the N and C-domains of CLIC2, would stabilize the protein in solution. Consequently, this increase in stability is hypothesized to lock the CLIC2 at the hinge region, thereby preventing insertion of the N-domain into the bilayer. In vitro experiments were conducted on the identified missense mutations in CLIC2 and compared to the wild type so as to assess the modeling predictions. In particular, the CLIC2 thermal stability and membrane binding properties were examined.

Using mutagenesis, the missense mutants were introduced into the recombinant CLIC2 protein. Circular dichroism (CD) and differential scanning fluorimetry (DSF) were conducted on the purified proteins to analyze their structural and thermal stability. The nsSNP’s showed no differences in secondary structure or thermal stability by CD or DSF, whereas the XLID linked mutant H101Q showed a 6^oC increase in stability as measured by DSF. Current studies, including Impedance Spectroscopy, are being performed to further investigate the binding interaction of the CLIC2 constructs with the lipid bilayer.