1H NMR studies of porcine calbindin D9k in solution: sequential resonance assignment, secondary structure, and global fold.
1H NMR studies of porcine calbindin D9k in solution: sequential resonance assignment, secondary structure, and global fold.
复制标题
溶液中猪钙结合蛋白 D9k 的 1 H NMR 研究：顺序共振分配、二级结构和整体折叠。
DOI：
10.1021/bi00440a035
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发表时间：
1989
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：
Drakenberg,T;Hofmann,T;Chazin,WJ
Torbjorn Drakenberg，** Theo Hofmann，11和Walter J. Chazin ** Scripps诊所研究所分子生物学系，10666 North Torrey Pines Road，拉霍亚，加州92037，和多伦多大学生物化学系，多伦多，安大略M5 S 1A 8，加拿大，1989年1月19日接收;修订后的Mandatapt，1989年3月31日接收摘要：对猪钙结合蛋白D9 k（78个氨基酸，Mr 8800）的~ 1H核磁共振谱进行了归属。超过98%的1H共振，包括每个氨基酸残基的自旋系统，已经通过使用整合来自广泛的二维标量相关NMR实验的数据的方法来鉴定[Chazin，Ranee，& Wright（1988）J. Mol. Biol. 202，603-626]。由于样品量的有限性和蛋白质构象的不均一性，二维核Overhauser效应（NOE）实验在自旋体系的确定中也起着重要的作用。根据标量连通性的模式，78个自旋系统中的43个可以直接指定为适当的剩余类型。这为获得序列特异性共振归属提供了充分的基础。二级结构的元素是从连续和中等范围的NOE，VNHa的值，和缓慢交换的骨架酰胺质子的位置确定的。溶液中存在四个明确定义的螺旋和两个钙结合环之间的迷你/3-折叠。这些二级结构元素和一些关键的长程NOE为确定蛋白质在溶液中的全局折叠提供了重要信息。通常，在牛钙结合蛋白D9 k的minorA形式的晶体结构和完整的猪钙结合蛋白D9 k的溶液结构之间发现良好的一致性。唯一的显著差异是牛晶体结构中螺旋II和III之间的环中有一个短的单圈螺旋，这在猪溶液结构中明显不存在。（钙结合蛋白D9 k（以前的肠钙结合蛋白）是一种小的、酸性的和非常热稳定的蛋白质，存在于迄今为止研究的所有哺乳动物物种的小肠中（Kallfelz等人，1967; Drescher & De Luca，1971; Hitchman & Harrison，1972; Taylor，1983）。它属于钙调节蛋白的肌钙蛋白C超家族，其特征在于钙离子通道的共同螺旋-环-螺旋基序。
Torbjorn Drakenberg,**'§ Theo Hofmann, 11 and Walter J. Chazin*’* Department of Molecular Biology, Research Institute of Scripps Clinic, 10666 North Torrey Pines Road, La Jolla, California 92037, and Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada Received January 19, 1989; Revised Manuscript Received March 31, 1989 abstract: The'H nuclear magnetic resonance(NMR) spectrum of Ca2+-saturated porcine calbindin D9k (78 amino acids, Mr 8800) has been assigned. Greater than 98% of the'H resonances, including spin systems for each amino acid residue, have been identified by using an approach that integrates data from a wide range of two-dimensional scalar correlated NMR experiments [Chazin, Ranee, & Wright (1988) J. Mol. Biol. 202, 603-626]. Dueto the limited quantity of sample and conformational heterogeneity of the protein, two-dimensional nuclear Overhauser effect (NOE) experiments also played an essential role in the iden-tification of spin systems. On the basis of the pattern of scalar connectivities, 43 of the 78 spin systems could be directly assigned to the appropriate residue type. This provided an ample basis for obtaining the sequence-specific resonance assignments. The elements of secondary structure are identified from sequential and medium-range NOEs, values of VNHa, and thelocation of slowly exchanging backbone amide protons. Four well-defined helices and a mini/3-sheet between the two calcium binding loops are present in solution. These elements of secondary structure and a few key long-range NOEs provided sufficientinformation to define the global fold of the protein in solution. Generally good agreement is found between the crystal structure of the minorA form of bovine calbindin D9k and the solution structure of intact porcine calbindin D9k. The only significant difference is a short one-turn helix in the loop between helices II and III in the bovine crystal structure, which is clearly absent in the porcine solution structure.(Calbindin D9k (formerly intestinal calcium binding protein) is a small, acidic, and very heat stable protein found in the small intestine of all mammalian species so far examined (Kallfelz et al., 1967; Drescher & De Luca, 1971; Hitchman & Harrison, 1972; Taylor, 1983). It belongs to the troponin C superfamily of calcium regulatory proteins that are char-acterized by a common helix-loop-helix motif for the calcium