Nuclear magnetic resonance spectroscopy of a DNA complex with the uniformly 13C-labeled Antennapedia homeodomain and structure determination of the DNA-bound homeodomain.
Nuclear magnetic resonance spectroscopy of a DNA complex with the uniformly 13C-labeled Antennapedia homeodomain and structure determination of the DNA-bound homeodomain.
复制标题
具有统一 13C 标记触角足同源域的 DNA 复合物的核磁共振波谱以及 DNA 结合同源域的结构测定。
DOI：
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发表时间：
1993
影响因子：
5.6
通讯作者：
K. Wüthrich
中科院分区：
文献类型：
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作者：
Y. Qian;G. Otting;M. Billeter;M. Müller;W. Gehring;K. Wüthrich
制备了由突变型触角足蛋白（C39S）同源结构域和一个14碱基对的DNA双链体（分子量约18000）形成的1∶1复合物，其中蛋白质被均匀地进行了13C标记。利用具有13C（ω1，ω2） - 双半滤波的二维核奥弗豪泽增强（NOE）光谱学以及三维13C相关NOE光谱学，获得了复合物中蛋白质和DNA几乎完整的序列特异性共振归属。在此基础上，收集了用于三维结构测定所需的构象限制条件。以855个分子内距离限制条件作为输入，用DIANA程序计算了与DNA结合的Antp（C39S）同源结构域的结构，随后用OPAL程序进行了受限能量最小化。一组20个构象异构体表征了残基8至56的明确结构，单个构象异构体的主链原子相对于平均坐标计算的成对均方根偏差平均为0.5 Å。所得结构的质量与游离蛋白质的结构相当，并且发现游离的Antp（C39S）同源结构域的整体折叠在DNA复合物中是保守的。与DNA结合的蛋白质的结构是随后测定溶液中完整的Antp（C39S）同源结构域 - DNA复合物结构的起点。
A 1:1 complex formed by the mutant Antennapedia(C39S) homeodomain and a 14 base-pair DNA duplex (molecular weight approximately 18,000) was prepared in which the protein was uniformly 13C-labeled. Using two-dimensional nuclear Overhauser enhancement (NOE) spectroscopy with 13C(omega 1, omega 2)-double-half-filter and three-dimensional 13C-correlated NOE spectroscopy, nearly complete sequence-specific resonance assignments were obtained for both the protein and the DNA in the complex. On this basis conformational constraints needed for a three-dimensional structure determination were collected. Using 855 intramolecular distance constraints as input, the structure of the DNA-bound Antp(C39S) homeodomain was calculated with the program DIANA, followed by restrained energy minimization with the program OPAL. A group of 20 conformers characterizes a well-defined structure for residues 8 to 56, with an average of 0.5 A of the pairwise root-mean-square deviations calculated for the backbone atoms of the individual conformers relative to the mean coordinates. The quality of the resulting structure is comparable to the one for the free protein, and the global fold of the free Antp(C39S) homeodomain was found to be conserved in the DNA complex. The structure of the DNA-bound protein was the starting point for the subsequent structure determination of the complete Antp(C39S) homeodomain-DNA complex in solution.