Solution structure, hydrodynamics and thermodynamics of the UvrB C-terminal domain.
Solution structure, hydrodynamics and thermodynamics of the UvrB C-terminal domain.
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DOI：
10.1080/07391102.2001.10506734
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发表时间：
2001-10-01
影响因子：
4.4
通讯作者：
Lane, AN
中科院分区：
文献类型：
--
作者：
Alexandrovich, A;Czisch, M;Lane, AN
用高分辨核磁共振、超速离心、N-15核磁共振弛豫、凝胶渗透、核磁共振扩散、圆二色谱和差示扫描量热法测定了UvrB的55个残基C-末端结构域的溶液结构、热力学稳定性和流体力学性质。亚基相对分子质量为7438 kDa，相对分子质量为14.5+/-1.0 kDa。由平衡沉积决定的，表示二聚体结构。由核磁共振确定的结构显示了一个稳定的反平行螺旋发夹的二聚体，以一种不寻常的方式结合在一起，具有一个小的疏水界面。蛋白质的斯托克斯半径从蛋白质浓度大于4微米时的高平台值(约22埃)减小到浓度小于0.1微米时的约18埃。远紫外圆二色谱的浓度和温度依赖性表明，该蛋白质是热稳定的(在36微米时的温度约为71.5℃)。与这些数据一致的最简单的模型是二聚体解离成折叠的单体，然后协同解开。通过拟合得到了这两种转变的范氏热焓和离解常数，其中DeltaH(1)约为23kJ mol(-1)，Ki(298)=0.4um，DeltaH(2)=184kJ mol(-1)。这与直接对蛋白质去折叠的量热分析是一致的，量热分析得到的热变为181kJ·mol(-1)，范氏热变为354kJ·mol(-1)，证实了二聚体到单体的展开。热力学数据可以与观察到的二聚化模式相一致。在14.1T和11.75T下的N-15核磁共振松弛测量证实，在mM浓度下，该蛋白质表现为不对称二聚体，与UvrB蛋白的其余部分连接有一个灵活的N-末端接头。该结构域的二聚化在切除修复机制中的作用被讨论。
The solution structure, thermodynamic stability and hydrodynamic properties of the 55-residue C-terminal domain of UvrB that interacts with UvrC during excision repair in E.coli have been determined using a combination of high resolution NMR, ultracentrifugation, N-15 NMR relaxation, gel permeation, NMR diffusion, circular dichroism and differential scanning calorimetry. The subunit molecular weight is 7,438 kDa., compared with 14.5 +/-1.0 kDa. determined by equilibrium sedimentation, indicating a dimeric structure. The structure determined from NMR showed a stable dimer of anti-parallel helical hairpins that associate in an unusual manner, with a small and hydrophobic interface. The Stokes radius of the protein decreases from a high plateau value (ca. 22 Angstrom) at protein concentrations greater than 4 muM to about 18 Angstrom at concentrations less than 0.1 muM. The concentration and temperature-dependence of the far UV circular dichroism show that the protein is thermally stable (T. ca. 71.5 degreesC at 36 muM). The simplest model consistent with these data was a dimer dissociating into folded monomers that then unfolds co-operatively. The van't Hoff enthalpy and dissociation constant for both transition was derived by fitting, with DeltaH(1 approximate to)23 kJ mol(-1), KI(298)=0.4 muM and DeltaH(2)=184 kJ mol(-1). This is in good agreement with direct calorimetric analysis of the then-nal unfolding of the protein, which gave a calorimetric enthalpy change of 181 kJ mol(-1) and a van't Hoff enthalpy change of 354 kJ mol(-1), confirming the dimer to monomer unfolding. The thermodynamic data can be reconciled with the observed mode of dimerisation. N-15 NMR relaxation measurements at 14.1 T and 11.75 T confirmed that the protein behaves as an asymmetric dimer at mM concentrations, with a flexible N-terminal linker for attachment to the remainder of the UvrB protein. The role of dimerisation of this domain in the excision repair mechanism is discussed.