蛋白质结构与功能NMR的研究.ppt

4. Ssh10b与核酸相互作用温度依赖性的结构基础 Ssh10b在核磁谱图上呈现两种象，比例 与温度相关．谱图分析表明两种构象是 L61-P62顺反异构造成的 L61-P62 反式 顺式 在新药发现中的NMR 应用 An important technique in support of structure-based drug design provide information on the nature of molecular interactions, identify weak-binding compounds to aid development of the drug-like inhibitors for use as lead compounds in drug discovery. NMR进行新药研究的策略 检测蛋白质： 1. 选择性地同位素标记某种类型的氨基酸残基; 2. ‘segmental labelling’: discrete segments of the polypeptide chain are uniformly labelled; 3. Chemical-shift mapping or Differential chemical-shift mapping 检测底物小分子： Based on T2 and T1enhancement: the large differences in the rates of rotational and translational motions of a small molecule in the free state relative to when it is bound to a macromolecule 新的脉冲方法： SEA-TROSY: selects solvent-exposed amide protons in uniformly deuterated 15N-labelled proteins dissolved in H2O by magnetization transfer from H2O. 二氢叶酸还原酶(DHPR) Met ε-13CH3 resonance region a. Chemical-shift mapping 方法： 绿色表示没有底物存在，蓝色表示存在底物类似物pyridine-2,6-dicarboxylate (PDC)． b. Differential chemical-shift mapping方法： 蓝色表示结合PDC，红色表示结合4-chloro-PDC. SEA-TROSY脉冲方法 Conventional [15N,1H]-HSQC [15N,1H]-TROSY SEA-TROSY selects solvent-exposed 1HN Methods based on T2 and T1enhancement 1. T2 (小分子) T2 (蛋白质) the resonance lines in the NMR spectra of small molecules are much narrower than those in the spectra of macromolecules. 2. T1 (小分子) transferred NOEs: small positive NOEs for free small ligand; negative NOEs for bound ligand. SAR (structure–activity relationship) by NMR SAR by NMR : use of protein chemical-shift changes to screen for low affinity ligands, and the collection of structural information to direct a linked-fragment approach to enhancing binding affinities. a,b. Small molecules that bind to two distinct sites on the target protein. A second screen of close analogues to the hit(s) can be used to optimize the affinity for the sites. c.