Cardiac-Specific Nkx2.5 Homeodomain: Conformational Stability and Specific DNA Binding of Nkx2.5(C56S)
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- 作者：Elfrieda Fodor ; James W. Mack ; Jin-Soo Maeng ; Jeong-Ho Ju ; Hyun Sook Lee ; James M. Gruschus ; James A. Ferretti ; and Ann Ginsburg
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：September 20, 2005
- 年：2005
- 卷：44
- 期：37
- 页码：12480 - 12490
- 全文大小：142K
- 年卷期：v.44,no.37(September 20, 2005)
- ISSN：1520-4995
文摘
The cardiac-specific Nkx2.5 homeodomain has been expressed as a 79-residue protein withthe oxidizable Cys56 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 × 108 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 Å2 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.
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 × 108 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 Å2 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.