1H NMR resonance assignments, secondary structure, and global fold of Apo bovine calbindin D9k.
1H NMR resonance assignments, secondary structure, and global fold of Apo bovine calbindin D9k.
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Apo 牛钙结合蛋白 D9k 的 1 H NMR 共振归属、二级结构和整体折叠。
DOI：
10.1021/bi00476a016
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发表时间：
1990
期刊：
影响因子：
2.9
通讯作者：
Chazin,WJ
中科院分区：
文献类型：
--
作者：
Skelton,NJ;Forsén,S;Chazin,WJ
用二维核磁共振氢谱研究了脱辅基牛钙结合蛋白D9 k的溶液结构和动力学。由于野生型蛋白中存在构象异质性，因此对Pro43-*· Gly突变体进行了序列共振归属。通过使用从H2O溶液获得的标量相关实验的组合，76 'H自旋系统中的61个可以被指定为特定的氨基酸类型。剩余的共振通过从2* H2O溶液中获得的一系列平行实验来分配。这些自旋系统的分配提供了一个完整的序列共振分配的基础，从残基间骨架核Overhauser效应（NOE）。二级结构的元素被确定从顺序和中程NOE，骨干自旋-自旋耦合常数，和缓慢交换酰胺质子。四个部分的螺旋划定，连同一个短的反平行/3-片层之间的相互作用的肽环参与钙离子结合。通过将二级结构的这些元素与长程螺旋间NOE的子集相结合来提供全局折叠。与对Ca 2+饱和蛋白的类似研究的比较表明，在该粗水平下，结构非常相似。然而，去除的Ca 2+显着影响蛋白质的动力学，酰胺质子交换率和芳环旋转判断。这在疏水核中残基的柔性增加中特别明显。（钙离子参与调节细胞代谢和功能的许多方面（Rasmussen，1986 a，B，1989）。外界刺激可引起胞浆内Ca ~（2+）浓度的瞬时升高，这些离子在刺激过程中充当第二信使。在许多情况下，Ca 2+信号由调节性Ca 2+结合蛋白翻译。在细胞刺激时，Ca 2+浓度（从静息细胞中的10 - 7 M水平）增加10-100倍足以引起这些蛋白质中的调节位点结合钙离子。已经提出，Ca 2+的结合引起蛋白质构象的变化，从而引发进一步的细胞再分化。
The solution structure and dynamics of apo bovine calbindin D9k have been studied by a wide range of two-dimensional’H nuclear magnetic resonance experiments. Due to the presence of conformational heterogeneity in the wild-type protein, the sequential resonance assignment was carried out on a Pro43-*• Gly mutant. By use of a combination of scalar correlation experiments acquired from H20 solution, 61 of the 76’H spin systems could be assigned to particular aminoacid types. The remaining resonances were assigned by a parallel series of experiments acquired from 2**** H20 solution. These spin system assignments provided a basis for complete sequential resonance assignments from interresidue backbone nuclear Overhauser effects (NOEs). Elements of secondary structure were identified from sequential and medium-range NOEs, backbone spin-spin coupling constants, and slowly exchanging amide protons. Four sections of helix are delineated, together with a short antiparallel/3-sheet interaction between the peptide loops involved in Ca2+ binding. The global fold is provided by combining these elements of secondary structure with a subset of the long-range, interhelix NOEs. Comparison with similar studies on the Ca2+-saturated protein indicates that at this crude level the structures are very similar. However, removal of the Ca2+ does dramatically affect the dynamics of the protein, as judged by amide protonexchange rates and aromatic ring rotation. This is particularly evident in the increased flexibility of the residues in the hydrophobic core.(Calcium ions are involved in the regulationof many aspects of cell metabolism and function (Rasmussen, 1986a, b, 1989). External stimuli lead totransient increases in cytoplasmic Ca2+ concentration, these ions acting as a secondary messenger in the stimultion process. In many cases, the Ca2+ signal is translated by regulatory Ca2+-binding proteins. The 10-100-fold increase inCa2+ concentration (from the 10 “7 M level in the resting cell) upon cell stimulation is sufficient to cause the regulatory sites in these proteins to bind calcium ions. It has been proposed that the binding of Ca2+ causes changes in the protein conformation that trigger further cellular re-