Backbone dynamics of calcium-loaded calbindin D9k studied by two-dimensional proton-detected 15N NMR spectroscopy.
Backbone dynamics of calcium-loaded calbindin D9k studied by two-dimensional proton-detected 15N NMR spectroscopy.
复制标题
通过二维质子检测 15N NMR 光谱研究负载钙的钙结合蛋白 D9k 的主链动力学。
DOI：
10.1021/bi00135a017
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发表时间：
1992
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：
Kördel,J;Skelton,NJ;Akke,M;Palmer3rd,AG;Chazin,WJ
斯克里普斯研究所分子生物系，加州拉霍亚92037摘要：利用二维质子检测的异核磁共振波谱技术，研究了负载钙的钙结合蛋白D9k的主链动力学。测定了72个主酰胺15N核中的71个的自旋-晶格弛豫速率常数、自旋-自旋弛豫速率常数和稳态j'Hf-^ N核Overhauser效应。使用无模型的形式分析弛豫参数，包括分子的整体旋转相关时间、广义序参数（S2）和每个酰胺基团的有效内相关时间。Calbindin D9k包含两个螺旋-环-螺旋基元，在蛋白的一端由一个连接环连接，在另一端两个钙结合环之间有一个/3型相互作用。从S2的平均值分别为0.83 ~ 0.05和0.85 ~ 0.04判断，钙结合环和螺旋的运动幅度相似。连接分子两个钙结合亚域的连接子区域具有明显更高的柔韧性，其平均S2值明显较低，为0.59 ~ 0.23。对于连接环和c端残基，序参量进一步分解为两个不同时间尺度上运动过程的单独序参量。螺旋I和IV的有效相关时间明显长于螺旋II和III或钙结合环。残基间的比较揭示了序参量与晶体学b因子和酰胺质子交换率的相关性，尽管这些性质在敏感的时间尺度上存在巨大差异。序参量还用于区分calbinding蛋白D9k的核磁共振衍生三维结构中由于固有的高柔韧性而定义较差的区域，以及具有平均柔韧性但结构约束密度较低的定义较差的区域。Calbindin D9k是一种小的钙结合蛋白，在结构上与钙调蛋白（calmodulin, CaM） 1和肌钙蛋白C （troponin C, TnC）的球状结构域同源。这些蛋白属于超家族蛋白，通过一个由两个螺旋组成的钙结合环组成的共同结构基元结合Ca2+离子，称为EF-hand （Kretsinger, 1987）。ef手通常成对出现，如图1所示为平行排列。在CaM超家族的一些成员中已经观察到钙结合的协同性，这被归因于两个钙结合环之间的/3型相互作用（Seamon & Kretsinger, 1983; Linse et al., 1987）。已经提出Ca2+离子的结合会导致螺旋的大量结构重排（Herzberg et al., 1986），这改变了性质
Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037 Received December 16, 1991; Revised Manuscript Received March 11, 1992 abstract: Backbone dynamics of calcium-loaded calbindin D9k have been investigated by two-dimensional proton-detected heteronuclear nuclear magnetic resonance spectroscopy, using a uniformly 15N enriched protein sample. Spin-lattice relaxation rateconstants, spin-spin relaxation rateconstants, and steady-state j'Hf-^ N nuclear Overhauser effects were determined for 71 of the 72 backbone amide 15N nuclei. The relaxation parameters were analyzed using a model-free formalism that incorporates the overall rotational correlation time of the molecule, and a generalized order parameter (S2) and an effective internal correlation time for each amide group. Calbindin D9k contains two helix-loop-helix motifs joined by a linker loop at one end of the protein and a/3-type interaction between the two calcium-binding loops at the other end. The amplitude of motions for the calcium-binding loops and the helices are similar, as judged from the average S2 values of 0.83 ą 0.05 and 0.85 ą 0.04, respectively. The linker region joining the two calcium-binding subdomains of the molecule has a significantly higher flexibility, as indicated by a substantially lower average S2 value of 0.59 ą 0.23. For residues in the linker loop and at the C-terminus, the order parameter is further decomposed into separate order parameters for motional processes on two distinct time scales. The effective correlation times are significantly longer for helices I and IV than for helices II and III or for the calcium-binding loops. Residue by residue comparisons reveal correlations of the order parameters with both the crystallographic B-factors and amide proton exchange rates, despite vast differences in the time scales to which these properties are sensitive. The order parameters are also utilized to distinguish regions of the NMR-derived three-dimensional structure of calbindin D9k that are poorly defineddue to inherently high flexibility, frompoorly definedregions with average flexibility but a low density of structural constraints.(Calbindin D9k is a small calcium-binding protein structurally homologous to the globular domains of calmodulin (CaM) 1 and troponin C (TnC). These proteins belong to the super-family of proteins that bind Ca2+ ions through a common structural motif consisting of a calcium-binding loop flanked by two helices, termed the EF-hand (Kretsinger, 1987). The EF-hands generally occur in pairs, with a parallel arrangement as detailed in Figure 1. Cooperativity in the binding of calcium has been observed for some members of the CaM superfamily and has been attributed to the/3-type interaction between the two calcium-binding loops (Seamon & Kretsinger, 1983; Linse et al., 1987). The binding of Ca2+ ions has been proposed to resultin substantial structural rearrangements of the helices (Herzberg et al., 1986), which changes the nature