Two-dimensional NMR studies of staphylococcal nuclease. 2. Sequence-specific assignments of carbon-13 and nitrogen-15 signals from the nuclease H124L-thymidine 3',5'-bisphosphate-Ca2+ ternary complex.
Two-dimensional NMR studies of staphylococcal nuclease. 2. Sequence-specific assignments of carbon-13 and nitrogen-15 signals from the nuclease H124L-thymidine 3',5'-bisphosphate-Ca2+ ternary complex.
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葡萄球菌核酸酶的二维核磁共振研究。
DOI：
10.1021/bi00453a012
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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;Hinck,AP;Loh,SN;Markley,JL
葡萄球菌核酸酶124 L样品（在大肠杆菌中过量产生的克隆蛋白，其序列与从金黄色葡萄球菌V8菌株分离的核酸酶的序列相同）用碳-13均匀标记（26% ul 13C），用氮气-15（95%ul 15N），并且具体地通过掺入氮-15-标记的亮氨酸（[98%15N] Leu）或碳-13-标记的赖氨酸（[26%ul 13C] Lys）、精氨酸（[26%ul 13C] Arg）或甲硫氨酸（[26%ul 13C] Met）。这些类似物（核酸酶124L-pdTp-Ca2+）的三元复合物在pH 5.1（H2O）或pH * 5.5（2H2O）和45 ℃下的溶液通过多核二维（2D）NMR实验在14.09和11.74 T的光谱仪场下分析标记图案：* H-13C单键相关（! H [13 C} SBC）;* H-13 C单键与NOE中继的相关性（* H {13 C} SBC-NOE）;* H-13 C单键与Hartmann-Hahn中继的相关性（* H-13 C SBC-NOE）;| I3c| SBC-HH）;* H-15 N单键相关性与NOE中继（'H {15 N} SBC-NOE）。这些结果有助于自旋系统的归属和二级结构元素的鉴定。在用13 C均匀标记的核酸酶三元复合物的1H（13 C）-SBC-NOE光谱中观察到反平行/3-折叠（daa NOE）的核Overhauser增强（NOE）特征。在 * H中观察到NOE的-螺旋特征（i/NN NOE）|15n|由15N均匀标记的蛋白质制备的复合物的SBC-NOE光谱。从这些多核NMR研究中获得的归属已经基于1H {1H} 2D NMR实验确认和扩展了归属[Wang，J.，LeMaster，D. M.，& Markley，J. L.葡萄球菌核酸酶作为研究结构-功能关系的模型系统已经研究了多年（Taniuchi & Anfinsen，1967; Tucker et al.，1978，1979; Evans等人，1987; Hibler等人，1987; Grissom & Markley，1989）。详细的NMR 1解决方案的抑制剂结合的影响，构象流动性，和蛋白质折叠的机制的研究需要广泛的光谱分配。在核酸酶的ID NMR谱中分配特定共振的早期尝试利用选择性氘代葡萄球菌核酸酶（Markley et al.，1978; Jardetzky等人，1972）和选择性硝化的核酸酶（Cohen等人，1971年）。组氨酸 * 1共振已经通过使用定点诱变在1D 1H NMR谱中指定（Alexandrescu等人，1988年）。
Samples of staphylococcal nuclease 124L (cloned protein overproduced in Escherichia coli whose sequence is identical with that of the nuclease isolated from the V8 strain of Staphylococcus aureus) were labeled uniformly with carbon-13 (26% ul 13C), uniformly with nitrogen-15 (95% ul 15N), and specifically by incorporating nitrogen-15-labeled leucine ([98% 15N] Leu) or carbon-13-labeled lysine ([26% ul 13C] Lys), arginine ([26% ul 13C] Arg), or methionine ([26% ul 13C] Met). Solutions of the ternary complexes of these analogues (nuclease 124L-pdTp-Ca2+) at pH 5.1 (H20) or pH* 5.5 (2H20) at 45 C were analyzed as appropriate to the labeling pattern by multinuclear two-dimensional (2D) NMR experiments at spectrometer fields of 14.09 and 11.74 T:* H-13C single-bond correlation (! H [13C} SBC);* H-13C single-bond correlation with NOE relay (* H {13C} SBC-NOE);* H-13C single-bond correlation with Hartmann-Hahn relay (* H-| I3C| SBC-HH);'H-13C multiple-bond correlation ('H) 13C) MBC);'H-15N single-bond correlation (‘H-j15N) SBC);* H-15N single-bond correlationwith NOE relay (’H {15N} SBC-NOE). Theresults have assisted in spin system assignments and in identification of secondary structural elements. Nuclear Overhauser enhancements (NOE’s) characteristic of antiparallel/3-sheet (daa NOE’s) were observed in the ‘H {13Cj-SBC-NOE spectrum of the nuclease ternary complexlabeled uniformly with 13C. NOE’s characteristic of-helix (i/NN NOE’s) were observed in the* H| 15N| SBC-NOE spectrum of the complex prepared from protein labeled uniformly with 15N. The assignments obtained from these multinuclear NMR studies have confirmed and extended assignments based on 1H {1H} 2D NMR experiments [Wang, J., LeMaster, D. M., & Markley, J. L.(1990) Biochemistry (preceding paper in this issue)].Staphylococcal nuclease has been studiedfor many years as a model system for investigations of structure-function relationships (Taniuchi & Anfinsen, 1967; Tucker et al., 1978, 1979; Evans et al., 1987; Hibler et al., 1987; Grissom & Markley, 1989). Detailed NMR1 solution studies of the effects of inhibitor binding, conformational mobility, and mechanisms of protein folding require extensive spectral assignments. An early attempt toassign specific resonances in the ID NMR spectrum of nuclease utilized selectively deuterated staphylococcal nuclease (Markley et al., 1978; Jardetzky et al., 1972) and selectivelynitrated nuclease (Cohen et al., 1971). Histidine* 1 resonances have been assigned in ID ‘H NMR spectra by using site-directed mutagenesis (Alexandrescu et al., 1988).
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影响因子：
2.9
作者：
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通讯作者：
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DOI：
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发表时间：
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期刊：
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影响因子：
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作者：
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影响因子：
2.9
作者：
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通讯作者：
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影响因子：
2.9
作者：
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影响因子：
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作者：
K. Shon;S. Opella
通讯作者：
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