Structural characterization of wildtype and Δ6-COOH NBD2.

<p>To evaluate potential changes in ABCC6 NBD2 resulting from the C-terminal deletion, NBD2 was expressed and purified for <i>in vitro</i> analysis. <i>A,</i> CD spectroscopy was used to evaluate changes in the secondary structure of the mutant NBD2. Spectra were collected from 260 to 198 nm and corrected for buffer absorbance. The traces were smoothed using a window of 5 nm. The wildtype NBD2, <i>black circles</i>, shows a mixed α/β secondary structure qualitatively consistent with known structures of NBD proteins. The Δ6-COOH mutant NBD2, <i>open circles</i>, shows no significant differences in CD spectra. <i>B,</i> analytical gel filtration was used to evaluate changes in hydrodynamic radii of the wildtype and mutant NBD2 proteins. The wildtype protein eluted as a single symmetrical peak at ∼12.2 mls, consistent with a protein of ∼25,000 Da MW. The mutant proteins eluted similarly, with a peak at 12.2 mls. No discernible differences in either CD or GFC could be detected between the wildtype and mutant proteins.