Cardiac-Specific Nkx2.5 Homeodomain:  Conformational Stability and Specific DNA Binding of Nkx2.5(C56S)

• Published in: Biochemistry (Easton) 2005-09, Vol.44 (37), p.12480-12490
• Main Authors: Fodor, Elfrieda, Mack, James W, Maeng, Jin-Soo, Ju, Jeong-Ho, Lee, Hyun Sook, Gruschus, James M, Ferretti, James A, Ginsburg, Ann
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino Acid Substitution; Animals; Binding Sites; Calorimetry; DNA - metabolism; Drosophila; Drosophila Proteins - chemistry; Drosophila Proteins - metabolism; Homeodomain Proteins - chemistry; Homeodomain Proteins - metabolism; Mice; Molecular Sequence Data; Mutagenesis, Site-Directed; Myocardium - metabolism; Protein Conformation; Protein Denaturation; Protein Structure, Secondary; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Restriction Mapping; Thermodynamics; Transcription Factors - chemistry; Transcription Factors - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi050835s

The cardiac-specific Nkx2.5 homeodomain has been expressed as a 79-residue protein with the oxidizable Cys56 replaced with Ser. The Nkx2.5 or Nkx2.5(C56S) homeodomain is 73% identical in sequence to and has the same NMR structure as the vnd (ventral nervous system defective)/NK-2 homeodomain 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 differential scanning calorimetry (DSC) and far-UV circular dichroism. Adding 100 mM NaCl to Nkx2.5(C56S) at pH 7.4 increases T m from 44 to 54 ± 0.2 °C and ΔH from 34 to 45 ± 2 kcal/mol (giving a ΔCp of ∼1.2 kcal K-1 mol-1 for homeodomain unfolding). DSC profiles of Nkx2.5 indicate fluctuating nativelike structures at