Analysis of three-dimensional structure of ion channel peptides bound to phospholipid bilayers by high resolution solid state NMR

通过高分辨率固态核磁共振分析与磷脂双层结合的离子通道肽的三维结构

• 批准号: 09640612
• 负责人: NAITO Akira
• 金额: $ 2.37万
• 依托单位: Himeji Institute of Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09640612/
• 关键词: Ion channel; Enkephalin; Melittin; Interatomic distance; High resolution solid state NMR; Three-dimensional structure; Phospholipid bilayer; Magnetically oriented membrane; アラメシチン; バイセル; イオンチャンネル; 膜分断; 膜融合

In this research project, we have studied to achieve a new approach to determine the three-dimensional structure and orientation of membrane bound ion channel peptides. 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) Interatomic distances of isotopically doubly labeled N-Acetyl-Pro-Gly-Phe crystals, obtained from rotational echo double resonance (REDOR) experiments, were analyzed as a three-spin system in which observed nuclei are simultaneously coupled with isotopically labeled intra- and intermolecular heteronuclei. In particular, the intermolecular dipolar interaction was examined to obtain a clue as to the relative orientation of a given molecule to the surrounding neighbors. We have developed a systematic procedure to determine both the intra- and intermolecular interatomic distances and the angle between three two interatomic vectors.(3) 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 bilayers. Finally, it was found that melittin adopts a transmembrane a-helix whose average axis is parallel to the bilayer normal.

本研究旨在实现一种新的方法来确定膜结合离子通道肽的三维结构和取向。本研究取得了以下成果。(1)用旋转回波双共振（REDOR）方法精确测定了六组[酪氨酸D113]-[甘氨酸D115]-[甘氨酸D11N]-亮氨酸脑啡肽（tyr-Gly-Gly-Phe-Leu）二水合物晶体的原子间距，并结合酪氨酸D113]-[甘氨酸D11C]-[甘氨酸D115]-[甘氨酸D1N]-[甘氨酸D1C]-[甘氨酸D1N]-[甘氨酸D1N]-[亮氨酸D1N]-[甘氨酸D1C]-[甘氨酸D1N]-[甘氨酸D1N]-[甘氨酸D1C]-[甘氨酸D1N]-[甘氨酸D1N]-[亮氨酸D1N]-[甘氨酸D1N]-[甘氨酸D1C]-[甘氨酸D1N]-[甘氨酸D1N]-[甘氨酸D1N]-[甘氨酸D1C]-[甘氨酸D1N]-[甘氨酸D1N]-[甘氨酸D1N]-[甘氨酸D1N]-[甘氨酸D1 (2)同位素双标记的N-乙酰基-Pro-Gly-Phe晶体，从旋转回波双共振（REDOR）实验获得的原子间距离进行了分析，作为一个三自旋系统中，所观察到的核同时耦合同位素标记的分子内和分子间的异核。特别是，分子间的偶极相互作用进行了检查，以获得一个线索，一个给定的分子周围的邻居的相对取向。我们已经开发了一个系统的程序来确定内部和分子间的原子间距离和三个两个原子间的矢量之间的角度。(3)用固体核磁共振技术研究了蜂毒肽与二肉豆蔻酰磷脂酰胆碱（DMPC）双层膜结合的构象和动力学以及在脂双层系统中的磁取向。利用双波长D131核磁共振（NMR）研究发现，在液晶-凝胶相变温度以上，蜂毒肽-DMPC双层膜体系形成了长轴平行于磁场的磁性取向的细长囊泡。在与脂质双层结合的蜂毒肽标记的蜂毒肽上观察到了β-D113 β-D1C 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。

项目成果

M. Kamihira: "A High-resolution Solid-state ^<13>C and ^<15>NMNR Study on Crystalline Leu-and Met-enkephalins : Distinction lf Polymorohs, Backbone Dynamics and Local Conformational Rearrangements Induced by Dehydration or Freezing Motions of Bound Solven

M. Kamihira：“结晶 Leu 和 Met 脑啡肽的高分辨率固态 ^<13>C 和 ^<15>NMNR 研究：Polymorohs 的区别、骨架动力学和由脱水或冷冻运动引起的局部构象重排

M. Tanio: "Evidence of Local Conformational Fluctuations and Changes in Bacteriorhodopsin, Dependent on Lipids, Detergents and Tremeric Structure, as Studied by^<13>CNMR"Biochim. Biophys. Acta. 1375. 84-92 (1998)

M. Tanio：“细菌视紫红质的局部构象波动和变化的证据，依赖于脂质、洗涤剂和Tremeric结构，如13 CNMR所研究的”Biochim。

S.Tuzi: "Location of a cation-binding site in the loop between helices F and G of bacteriorhodopsin as studied by ^<13>C NMR"Biophys.J.. 76. 1523-1531 (1999)

S.Tuzi：“通过 13 C NMR 研究细菌视紫红质螺旋 F 和 G 之间环中阳离子结合位点的位置”Biophys.J.. 76. 1523-1531 (1999)

A.Naito: "Backbone Dynamics of Polycrystalline Peptides Studied by Measurements of 15N NMR Lineshpes and 13C Transverse Relaxation Times" J.Mol.Structure. 441. 231-242 (1998)

A.Naito：“通过测量 15N NMR 谱线和 13C 横向弛豫时间研究多晶肽的骨架动力学”J.Mol.Structure。

S. Nakatsuji: "Preparation and prorerties of 2-(o-halophenyl)-a-nitonyl nitroxides"Mol. Cryst. Liq. Cryst.. 306. 279-284 (1997)

S. Nakatsuji：“2-(邻卤苯基)-α-硝基硝基氧的制备和性质”Mol。