Two-dimensional NMR studies of staphylococcal nuclease. 1. Sequence-specific assignments of hydrogen-1 signals and solution structure of the nuclease H124L-thymidine 3',5'-bisphosphate-Ca2+ ternary complex.
Two-dimensional NMR studies of staphylococcal nuclease. 1. Sequence-specific assignments of hydrogen-1 signals and solution structure of the nuclease H124L-thymidine 3',5'-bisphosphate-Ca2+ ternary complex.
复制标题
葡萄球菌核酸酶的二维核磁共振研究。
DOI：
10.1021/bi00453a011
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发表时间：
1990
期刊：
影响因子：
2.9
通讯作者：
Markley,JL
中科院分区：
文献类型：
--
作者：
Wang,JF;LeMaster,DM;Markley,JL
葡萄球菌核酸酶H124 L是在大肠杆菌中产生的重组蛋白，其序列与由金黄色葡萄球菌V8变体产生的核酸酶的序列相同。用二维核磁共振技术研究了酶-金属离子激活剂-核苷酸抑制剂三元复合物--核酸酶H124 L-胸苷3/，5/-二磷酸-Ca ~（2+）。酶的高效过量生产促进了随机部分氘代蛋白质的产生，这被证明是详细的NMR分析所必需的。1H NMR自旋系统通过常规的2D“Hj”Hl方法分析：COSY、中继COSY、HOHAHA和NOESY。通过NMR实验获得的NMR谱通过 * H-13 C和 * H-15 N杂原子NMR实验确认和扩展[Wang，J.，Hinck，A. P.，洛，S. N.，& Markley，J. L.（1990）生物化学（以下文件在这个问题）]。在自然丰度和50%随机分数氘代蛋白质制备的三元复合物的光谱提供了149个氨基酸残基中的121个的序列特异性分配所需的信息。短程和中程NOE连通性允许的三元复合物的二级结构特征的测定：三螺旋域和三个反平行/3折叠片与几个反向转弯。一些非连续的长程HN-HN和H“-HN连接性揭示了这些二级结构元件的空间排列的额外信息。该三元络合物的溶液结构显示出密切对应的3 '，5，-二磷酸-Ca ~（2+）三元络合物[Cotton，
Staphylococcal nuclease H124L is a recombinant protein produced in Escherichia coli whose sequence is identical with that of the nuclease produced by the V8 variant of Staphylococcus aureus. The enzyme-metal ion activator-nucleotide inhibitor ternary complex, nuclease H124L-thymidine 3/, 5/-bisphosphate-Ca2+, was investigated by two-dimensional (2D) NMR techniques. Efficient overproduction of the enzyme facilitated the production of random fractionally deuterated protein, which proved essential for detailed NMR analysis.* H NMR spin systems were analyzed by conventional 2D'Hj’Hl methods: COSY, relayed COSY, HOHAHA, and NOESY. Assignments obtained by NMR experiments were confirmed and extended by* H-13C and* H-15N heteronuclear NMR experiments [Wang, J., Hinck, A. P., Loh, S. N., & Markley, J. L.(1990) Biochemistry (following paper in this issue)]. Spectra of the ternary complexes prepared with protein at natural abundance and at 50% random fractional deuteration provided the information needed for sequence-specific assignments of 121 of the 149 amino acid residues. Short-and intermediate-range NOE connectivities allowed the determination of secondary structural features of the ternary complex: three-helical domains and three antiparallel/3-pleated sheets with several reverse turns. A number of nonsequential long-range HN-HN and H “-HN connectivities revealed additional in-formation about the spatial arrangement of these secondary structural elements. The solution structure of this ternary complex shows a close corresponden 3', 5,-bisphosphate-Ca2+ ternary complex [Cotton,