The vnd/NK-2 homeodomain: thermodynamics of reversible unfolding and DNA binding for wild-type and with residue replacements H52R and H52R/T56W in helix III.

The conformational stabilities of the vnd (ventral nervous system defective)/NK-2 homeodomain [HD(wt); residues 1−80 that encompass the 60-residue homeodomain] and those harboring mutations in helix III of the DNA recognition site [HD(H52R) and HD(H52R/T56W)] have been investigated by differential scanning calorimetry (DSC) and ellipticity changes at 222 nm. Thermal unfolding reactions at pH 7.4 are reversible and repeatable in the presence of 50−500 mM NaCl with ΔCp = 0.52 ± 0.04 kcal K-1 mol-1. A substantial stabilization of HD(wt) is produced by 50 mM phosphate or by the addition of 100−500 mM NaCl to 50 mM Hepes, pH 7.4, buffer (from Tm = 35.5 °C to Tm 43−51 °C; ΔHvH ≅ 47 ± 5 kcal mol-1). The order of stability is HD(H52R/T56W) > HD(H52R) > HD(wt), irrespective of the anions present. Progress curves for ellipticity changes at 222 nm as a function of increasing temperature are fitted well by a two-state unfolding model, and the cooperativity of secondary structure changes is greater for mutant homeodom...