Expression and purification of the second homeodomain of the transcriptional regulator Zinc-finger homeobox 2.
TL;DR
The development of hypoalgesia does not seem to be related to global conformational changes in the structure of the domain, as monitored by those macroscopic probes.
Abstract
Zinc finger homeobox 2 (ZFHX2) is a 2572-residue-long, multidomain protein involved in the rare inherited congenital hypoalgesia. This pain insensitive phenotype is associated with a missense mutation (arginine to lysine at R1913) at the C-terminus of the second homeodomain (HDo2), comprising residues 1857-1916 of ZFHX2. In this work, we expressed and purified wild-type HDo2 and the mutant R1913K by using Ni2+-IMAC and a polishing gel filtration chromatography step. The isolated wild-type HDo2 was a monomeric, well-folded, stable polypeptide, with a high percentage of α-helix-like structure, as judged from the far-ultraviolet (UV) circular dichroism (CD), fluorescence and 1D-1H-NMR spectra. Similar conformational properties were observed for the R1913K mutant. Thermal denaturations for both species, followed by fluorescence and far-UV CD, yielded similar apparent thermal denaturation midpoints. Therefore, the development of hypoalgesia does not seem to be related to global conformational changes in the structure of the domain, as monitored by those macroscopic probes.