NUCLEAR MAGNETIC RESONANCE--NEW METHODS AND MOLECULAR STRUCTURE DETERMINATION

已开发出便于共振归属的核磁共振方法 对于可以用碳13和碳13进行同位素浓缩的蛋白质 N-15用于研究未标记的配体与 统一标记的蛋白质。将这些新方法应用于 蛋白质钙调素，在没有和存在非标记多肽的情况下 骨骼肌肌球蛋白轻链激酶片段，显示明显 钙调蛋白的结构差异。在有钙和In存在的情况下 缺少多肽，钙调蛋白由两个球状结构域组成， 由柔性链接器连接。氮-15松弛研究表明 这两个球状结构域重定向几乎是等规的，而不是 在结晶状态下观察到的结构应该是什么？ 其中两个结构域通过长的`-螺旋(所谓的 “中央螺旋”)。在肽存在的情况下，相对取向 这两个钙调蛋白结构域被很好地确定。多肽-蛋白质 复合体采用近似椭圆形的形状，其中的多肽 夹在两个结构域之间的`螺旋构象。这些结构 到目前为止的计算结果只基于光谱的一小部分 可从3D和4D核磁共振谱获得的信息。工作目前正在进行中 正在通过添加更多的NOE和J-联轴器来优化结构 约束条件。 使用脉冲场梯度的光谱。

NMR methods have been developed that facilitate the resonance assignment for proteins that can be isotopically enriched with carbon-13 and nitrogen-15 for the study of unlabeled ligands complexed with the uniformly labeled protein. Application of these new methods to the protein calmodulin, in the absence and presence of an unlabeled peptide fragment of skeletal muscle myosin light chain kinase, reveal pronounced structural differences in calmodulin. In the presence of calcium and in the absence of peptide, calmodulin consists of two globular domains, connected by a flexible linker. Nitrogen-15 relaxation studies indicate that the two globular domains reorient nearly isotopically, in contrast to what would be expected for the structure observed in the crystalline state where the two domains are connected by a long `-helix (the so-called "central helix"). In the presence of peptide, the relative orientation of the two calmodulin domains is well determined. The peptide-protein complex adopts an approximately ellipsoidal shape, with the peptide in an `-helical conformation clamped in between the two domains. The structures calculated to date are based on only a fraction of the spectral information available from the 3D and 4D NMR spectra. Work is currently in progress to refine the structure by adding more NOE and J-coupling constraints. spectra using pulsed field gradients.