Cardiac-specific Nkx2.5 homeodomain: Conformational stability and specific DNA binding of Nkx2.5(C56S)

E. Fodor, James W. Mack, Jin Soo Maeng, Jeong Ho Ju, Hyun Sook Lee, James M. Gruschus, James A. Ferretti, Ann Ginsburg

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

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 Tm 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 8 M-1 from 10 to 37 °C and a stoichiometry of 1:1 for homeodomain binding DNA, using isothermal titration calorimetry. The DNA binding reaction of Nkx2.5 is enthalpically controlled, and the temperature dependence of ΔH gives a ΔC p of -0.18 ± 0.01 kcal K-1 mol-1. This corresponds to 648 ± 36 Å2 of buried apolar surface upon Nkx2.5(C56S) binding duplex B-DNA. Thermodynamic parameters differ for Nkx2.5 and vnd/NK-2 homeodomains binding specific DNA. Unbound NK-2 is more flexible than Nkx2.5.

Original languageEnglish
Pages (from-to)12480-12490
Number of pages11
JournalBiochemistry
Volume44
Issue number37
DOIs
Publication statusPublished - Sep 20 2005

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DNA
Differential Scanning Calorimetry
Neurology
Nervous System
Differential scanning calorimetry
B-Form DNA
Calorimetry
Circular Dichroism
Titration
Thermodynamics
Stoichiometry
Drosophila
Hot Temperature
Nuclear magnetic resonance
Temperature
NK 2
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Fodor, E., Mack, J. W., Maeng, J. S., Ju, J. H., Lee, H. S., Gruschus, J. M., ... Ginsburg, A. (2005). Cardiac-specific Nkx2.5 homeodomain: Conformational stability and specific DNA binding of Nkx2.5(C56S). Biochemistry, 44(37), 12480-12490. https://doi.org/10.1021/bi050835s

Cardiac-specific Nkx2.5 homeodomain : Conformational stability and specific DNA binding of Nkx2.5(C56S). / Fodor, E.; Mack, James W.; Maeng, Jin Soo; Ju, Jeong Ho; Lee, Hyun Sook; Gruschus, James M.; Ferretti, James A.; Ginsburg, Ann.

In: Biochemistry, Vol. 44, No. 37, 20.09.2005, p. 12480-12490.

Research output: Contribution to journalArticle

Fodor, E, Mack, JW, Maeng, JS, Ju, JH, Lee, HS, Gruschus, JM, Ferretti, JA & Ginsburg, A 2005, 'Cardiac-specific Nkx2.5 homeodomain: Conformational stability and specific DNA binding of Nkx2.5(C56S)', Biochemistry, vol. 44, no. 37, pp. 12480-12490. https://doi.org/10.1021/bi050835s
Fodor, E. ; Mack, James W. ; Maeng, Jin Soo ; Ju, Jeong Ho ; Lee, Hyun Sook ; Gruschus, James M. ; Ferretti, James A. ; Ginsburg, Ann. / Cardiac-specific Nkx2.5 homeodomain : Conformational stability and specific DNA binding of Nkx2.5(C56S). In: Biochemistry. 2005 ; Vol. 44, No. 37. pp. 12480-12490.
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abstract = "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 Tm 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 8 M-1 from 10 to 37 °C and a stoichiometry of 1:1 for homeodomain binding DNA, using isothermal titration calorimetry. The DNA binding reaction of Nkx2.5 is enthalpically controlled, and the temperature dependence of ΔH gives a ΔC p of -0.18 ± 0.01 kcal K-1 mol-1. This corresponds to 648 ± 36 {\AA}2 of buried apolar surface upon Nkx2.5(C56S) binding duplex B-DNA. Thermodynamic parameters differ for Nkx2.5 and vnd/NK-2 homeodomains binding specific DNA. Unbound NK-2 is more flexible than Nkx2.5.",
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