Kördel J, Skelton N J, Akke M, Palmer A G, Chazin W J
Department of Molecular Biology, Scripps Research Institute, La Jolla, California 92037.
Biochemistry. 1992 May 26;31(20):4856-66. doi: 10.1021/bi00135a017.
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 rate constants, spin-spin relaxation rate constants, and steady-state [1H]-15N 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 beta-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 +/- 0.05 and 0.85 +/- 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 +/- 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 defined due to inherently high flexibility, from poorly defined regions with average flexibility but a low density of structural constraints.
利用均匀富集15N的蛋白质样品，通过二维质子检测异核核磁共振光谱研究了钙负载的钙结合蛋白D9k的主链动力学。测定了72个主链酰胺15N核中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[1H]-15N核Overhauser效应。使用无模型形式分析弛豫参数，该形式包含分子的整体旋转相关时间、每个酰胺基团的广义序参数（S2）和有效内部相关时间。钙结合蛋白D9k包含两个螺旋-环-螺旋基序，在蛋白质一端通过连接环连接，在另一端两个钙结合环之间存在β型相互作用。从平均S2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结构域的连接区域具有明显更高的灵活性，平均S2值显著更低，为0.59±0.23。对于连接环和C末端的残基，序参数进一步分解为两个不同时间尺度上运动过程的单独序参数。螺旋I和IV的有效相关时间比螺旋II和III或钙结合环的有效相关时间长得多。逐个残基比较揭示了序参数与晶体学B因子和酰胺质子交换率的相关性，尽管这些性质敏感的时间尺度存在巨大差异。序参数还用于区分钙结合蛋白D9k的NMR衍生三维结构中由于固有高灵活性而定义不明确的区域，与具有平均灵活性但结构约束密度低的定义不明确区域。
