The cardiac-specific Nkx2.5 homeodomain has been expressed as a 79-residue protein withthe oxidizable Cys
56 replaced with Ser. The Nkx2.5 or Nkx2.5(C56S) homeodomain is 73% identical insequence to and has the same NMR structure as the vnd (ventral nervous system defective)/NK-2homeodomain of
Drosophila when bound to the same specific DNA. The thermal unfolding of Nkx2.5(C56S) at pH 6.0 or 7.4 is a reversible, two-state process with unit cooperativity, as measured by differentialscanning calorimetry (DSC) and far-UV circular dichroism. Adding 100 mM NaCl to Nkx2.5(C56S) atpH 7.4 increases
Tm from 44 to 54 ± 0.2
C and
H from 34 to 45 ± 2 kcal/mol (giving a
Cp of ~1.2kcal K
-1 mol
-1 for homeodomain unfolding). DSC profiles of Nkx2.5 indicate fluctuating nativelikestructures at <37
C. Titrations of specific 18 bp DNA with Nkx2.5(C56S) in buffer at pH 7.4 with 100mM NaCl yield binding constants of 2-6 × 10
8 M
-1 from 10 to 37
C and a stoichiometry of 1:1 forhomeodomain binding DNA, using isothermal titration calorimetry. The DNA binding reaction of Nkx2.5is enthalpically controlled, and the temperature dependence of
H gives a
Cp of -0.18 ± 0.01 kcal K
-1mol
-1. This corresponds to 648 ± 36 Å
2 of buried apolar surface upon Nkx2.5(C56S) binding duplexB-DNA. Thermodynamic parameters differ for Nkx2.5 and vnd/NK-2 homeodomains binding specificDNA. Unbound NK-2 is more flexible than Nkx2.5.