Development of systematic approach in determining three-dimensional structure of membrane bound biomolecules based on accurate interatomic distances

在这个研究项目中，我们研究了建立系统方法，根据固态核磁共振获得的精确原子间距离来确定膜结合生物分子的三维结构。本研究项目取得了以下成果：(1)基于通过旋转回波双共振(REDOR)精确测定的六组D113D1C-D115D1N原子间距离以及D11C化学的一些附加约束，确定了[D113D1C，D115D1N]标记的Leu-脑啡肽(tyr-Gly-Gly-Phe-Leu)二水合物晶体的三维结构。 (2)使用固态NMR光谱检查了从四种溶剂生长的晶体中[D11D1C-D25D2]PheD14标记的LeuD15D1-和MetD1-D1-脑啡肽分子的苯环动力学。 NMR 粉末图案清楚地表明存在绕苯环 Cβ-Cγ 键轴的 180° 翻转运动。 (3)采用高分辨率固态核磁共振波谱研究了人降钙素(hCT)在酸性和中性条件下原纤维形成过程中的构象转变。通过固态 D131D1P 和 D113D1C NMR 光谱研究了蜂毒肽与二肉豆蔻酰磷脂酰胆碱 (DMPC) 双层结合的动力学以及脂质双层系统中的磁性取向。使用D131D1P NMR，发现蜂毒肽-DMPC双层系统在高于液晶-凝胶相变温度时形成磁性取向的细长囊泡，其长轴平行于磁场。在与脂质双层结合的 D113D1C 标记的蜂毒肽上观察到 D113D1C NMR 谱。最后发现蜂毒肽采用跨膜α螺旋，其平均轴平行于双层法线。较少的

In this research project, we have studied to establish systematic approach in determining the three-dimensional structure of membrane bound biomolecules based on accurate interatomic distances obtained from solid state NMR. Following results were obtained in this research project.(1) The three-dimensional structure of [ィイD113ィエD1C, ィイD115ィエD1N]-labeled Leu-enkephalin (tyr-Gly-Gly-Phe-Leu) dihydrate crystals was determined on the basis of six sets of accurately determined ィイD113ィエD1C-ィイD115ィエD1N interatomic distances by rotational echo double resonance (REDOR) and some additional constraints from ィイD113ィエD1C chemical shifts.(2) The phenyl ring dynamics of [ィイD12ィエD1HィイD25ィエD2]PheィイD14ィエD1-labeled LeuィイD15ィエD1- and MetィイD15ィエD1-enkephalin molecules in crystals grown from four solvents were examined using solid state ィイD12ィエD1H NMR spectroscopy. ィイD12ィエD1H NMR powder pattern clearly indicated the presence of 180°flip motions about the Cβ-Cγbond axis of the phenyl rings. The difference of … More the frequencies for the motion was attributed to the manner of their molecular packing in the crystal.(3) Conformational transition of human calcitonin (hCT) during fibril formation in the acidic and neutral condition were investigated by high resolution solid state ィイD113ィエD1C NMR spectroscopy. The results indicate that conformational transitions from α-helix to β-sheet, and from random coil to β-sheet forms occurred in the central and C-terminus regions, respectively, during fibril formation.(4) The conformation and dynamics of melittin bound to the dimyristoylphophatidylcholin (DMPC) bilayer and the magnetic orientation in the lipid bilayer systems were investigated by solid-state ィイD131ィエD1P and ィイD113ィエD1C NMR spectroscopy. Using ィイD131ィエD1P NMR, it was found that melittin-DMPC bilayer system forms magnetically oriented elongated vesicles with the long axis parallel to the magnetic field above the liquid crystalline-gel phase transition temperature. ィイD113ィエD1C NMR spectra were observed on the ィイD113ィエD1C labeled melittin bound to the lipid bilayer. Finally, it was found that melittin adopts a transmembrane α-helix whose average axis is parallel to the bilayers normal. Less