Fodor Elfrieda, Mack James W, Maeng Jin-Soo, Ju Jeong-Ho, Lee Hyun Sook, Gruschus James M, Ferretti James A, Ginsburg Ann
Section on Protein Chemistry, Laboratory of Biochemistry, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-8012, USA.
Biochemistry. 2005 Sep 20;44(37):12480-90. doi: 10.1021/bi050835s.
The cardiac-specific Nkx2.5 homeodomain has been expressed as a 79-residue protein with the oxidizable Cys(56) 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 degrees C and DeltaH from 34 to 45 +/- 2 kcal/mol (giving a DeltaC(p) of approximately 1.2 kcal K(-)(1) mol(-)(1) for homeodomain unfolding). DSC profiles of Nkx2.5 indicate fluctuating nativelike structures at <37 degrees C. Titrations of specific 18 bp DNA with Nkx2.5(C56S) in buffer at pH 7.4 with 100 mM NaCl yield binding constants of 2-6 x 10(8) M(-)(1) from 10 to 37 degrees 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 DeltaH gives a DeltaC(p) of -0.18 +/- 0.01 kcal K(-)(1) mol(-)(1). This corresponds to 648 +/- 36 A(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.
心脏特异性的Nkx2.5同源结构域已被表达为一种79个氨基酸残基的蛋白质，其中可氧化的半胱氨酸（Cys56）被丝氨酸取代。当与相同的特定DNA结合时，Nkx2.5或Nkx2.5（C56S）同源结构域与果蝇的vnd（腹侧神经系统缺陷）/NK-2同源结构域在序列上有73%的同一性，并且具有相同的核磁共振结构。通过差示扫描量热法（DSC）和远紫外圆二色性测量，在pH 6.0或7.4条件下，Nkx2.5（C56S）的热解折叠是一个具有单位协同性的可逆双态过程。在pH 7.4条件下，向Nkx2.5（C56S）中加入100 mM NaCl可使熔点（T(m)）从44℃升高到54±0.2℃，焓变（ΔH）从34 kcal/mol升高到45±2 kcal/mol（同源结构域解折叠的热容变化量（ΔC(p)）约为1.2 kcal K⁻¹ mol⁻¹）。Nkx2.5的DSC图谱表明在低于37℃时存在类似天然结构的波动。在pH 7.4且含有100 mM NaCl的缓冲液中，用Nkx2.5（C56S）滴定特定的18 bp DNA，通过等温滴定量热法得到10至37℃范围内的结合常数为2 - 6×10⁸ M⁻¹，同源结构域与DNA的结合化学计量比为1:1。Nkx2.5的DNA结合反应受焓控制，ΔH的温度依赖性给出的ΔC(p)为 -0.18±0.01 kcal K⁻¹ mol⁻¹。这对应于Nkx2.5（C56S）与双链B - DNA结合时648±36 Ų的埋藏非极性表面。Nkx2.5和vnd/NK - 2同源结构域结合特定DNA的热力学参数不同。未结合的NK - 2比Nkx2.5更具柔性。
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