Site-specific unfolding thermodynamics of a helix-turn-helix protein
Site-specific unfolding thermodynamics of a helix-turn-helix protein
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DOI：
10.1021/ja802185e
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发表时间：
2008-07-02
影响因子：
15
通讯作者：
Kubelka, Jan
中科院分区：
文献类型：
--
作者：
Amunson, Krista E.;Ackels, Loren;Kubelka, Jan
用圆二色谱（CD）和傅立叶变换红外光谱（FTIR）结合位点特异性碳13同位素标记研究了P22病毒外壳蛋白的40个残基螺旋-转角-螺旋亚结构域的热去折叠。螺旋-转角-螺旋是最简单的α-螺旋结构基序，它结合了二级和三级结构元件。个别螺旋片段的CD显示P22亚结构域通过三级螺旋间相互作用而稳定。总的来说，温度依赖性CD和FTIR数据可以通过具有部分折叠中间体的三态过程来描述。然而，位点特异性的C-13 IR信号的分析揭示了不同的展开热力学为每个j的标记位点。采用奇异值分解结合目标变换和全局拟合的方法得到了每个标记片段的热去折叠热力学参数。P22亚结构域从N-末端向转弯附近的螺旋段展开。我们的研究结果表明，只有两个C-13标记的残基可以在40个残基的蛋白质中检测到，并提供有关蛋白质展开的局部，位点特异性结构信息，这是不能解决的标准，非位点特异性光谱探针。
The thermal unfolding of a 40-residur helix-turn-helix subdomain of the P22 viral coat protein was investigated using circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) with site-specific C-13 isotopic labelling. Helix-turn-helix is the simplest a-helical structural motif that combines both secondary and tertiary structural elements. The CD of individual helical fragments reveals that the P22 subdomain is stabilized by tertiary interhelical interactions. Overall the temperature-dependent CD and FTIR data can be described by a three-state process with a partially folded intermediate. However, the analysis of the site-specific C-13 IR signals reveals distinct unfolding thermodynamics for each jof the labeled sites. The thermodynamic parameters of the thermal unfolding of each of the labeled segements were obtained using singular value decomposition in combination with target transformation and global fitting. The P22 subdomain unfolds from the N-terminus toward the helical segments near the turn. Our results show that as few as two C-13 labeled residues can be detected in a 40 residue protein and provide local, site-specific structural information about protein unfolding, which is not resolved by standard, nonsite-specific spectroscopic probes.