NUCLEAR MAGNETIC RESONANCE--NEW METHODS AND MOLECULAR STRUCTURE DETERMINATION

本文提出了一种新的核磁共振方法来测量三键J 联轴节.该方法适用于同位素富集的 大分子和浓缩溶液的分子在自然 丰饶。原子核之间的三键J耦合提供了非常 关于介入二面角的大小的直接信息，以及 从而在结构确定中产生有价值的新约束 生物大分子的过程。该方法已成功地 应用于13 C-13 C、13 C-15 N、1H-15 N、1H-13 C、1H-113 Cd、 和HN-Ha J偶联。 这是第一次证明，详细的结构， 洗涤剂溶解的蛋白质的分析是可能的， 全氘洗涤剂这种类型的分析依赖于组合使用 13 C和15 N编辑的NMR实验和脉冲场梯度， 使得可以抑制强烈的洗涤剂1H NMR信号， 观察到了弱得多的蛋白质信号。该方法被证明为 蛋白质钙调磷酸酶B，通过15摩尔过量的 洗涤剂CHAPS。钙调神经磷酸酶A/B复合物在细胞中保持完全活性。 CHAPS的存在和NMR结果证实洗涤剂不 显著改变蛋白质结构。谐振分配 主链1H、13 C和15 N共振和二级结构 确定过程已经完成。很明显，第二次 钙调神经磷酸酶B的结构与较小的 蛋白钙调素

A new NMR approach has been developed for the measurement of three-bond J couplings. The approach is applicable to isotopically enriched macromolecules and to concentrated solutions of molecules at natural abundance. The three-bond J couplings between atomic nuclei provide very direct information on the magnitude of the intervening dihedral angles and thereby yield valuable new constraints in the structure determination process of biological macromolecules. The approach has been successfully applied to the measurement of 13C-13C, 13C-15N, 1H-15N, 1H-13C, 1H-113Cd, and HN-Ha J couplings in a range of proteins. It has been demonstrated for the first time that a detailed structural analysis of a detergent-solubilized protein is possible without recourse to perdeuterated detergent. This type of analysis relies on the combined use of 13C- and 15N-edited NMR experiments and pulsed field gradients which make it possible to suppress the intense detergent 1H NMR signals while observing the much weaker protein signals. The approach is demonstrated for the protein calcineurin B, solubilized by a 15 molar excess of the detergent CHAPS. The calcineurin A/B complex remains fully active in the presence of CHAPS and NMR results confirm that the detergent does not significantly alter the protein structure. Resonance assignment of the backbone 1H,13C, and 15N resonances and the secondary structure determination process have been completed. It is evident that the secondary structure of calcineurin B is highly homologous with that of the smaller protein calmodulin.