Figure 8.

Biophysical characterization and limited proteolysis of the FLNC p.V930_T933del mutation. (A) Circular dichroism spectroscopy performed with purified, bacterially expressed wild-type and deletion mutant FLNC d7-8ΔVKYT constructs. The mean residue ellipticity (ΔεMR) of the wild-type shows a minimum in the spectrum at 218 nm and a positive value at 205 nm, while the deletion mutant displays a blue shift with minimum at 208 nm and positive ellipticity at 200 nm, a characteristic sign of the presence of unstructured regions. Solid line = wild-type; dashed line = FLNC d7-8ΔVKYT construct. (B) Differential scanning fluorimetry. Temperature denaturation experiments for FLNC d7–8 and deletion mutant FLNC d7–8ΔVKYT. The melting temperature (T_m) is determined at the inflection point of the fluorescence signal, the first derivative of which is reported here. Solid line = wild-type; dashed line = deletion mutant, scaled to the wild-type. (C) Protein stability analysed by thermolysin digestion. Wild-type and mutant FLNC d5-9ΔVKYT were treated with the protease thermolysin for 1 to 60 min, as indicated above each lane. Samples were analysed by PAGE. Note that the mutant protein was completely digested after 30 min, while a significant portion of the wild-type variant was still intact after 60 min of incubation, indicating less stable folding of the mutant protein. (D) Ribbon diagram of homology model of immunoglobulin-like domain 7 of human FLNC. β-strands B, E and D forming a β-sheet are coloured orange, green and yellow, respectively. Main chain atoms of residues involved hydrogen bonds indicated by dashed lines, stabilizing the β-sheet are depicted in stick model, with N and O coloured in blue and red. Residues 930VKYT933 that are missing in the deletion mutant are shown as full-atom stick model and coloured in red.