Mechanism of membrane fusion by fusion protein

本课题利用流感融合肽研究病毒感染的分子机制。我们特别关注于确定肽与膜结合的结构和方向。在本研究项目中获得了以下结果：(1)采用固态^<31>P和^<13>C核磁共振波谱法研究了蜂鸟素与二肉豆科磷脂（DMPC）双分子层结合的构象和动力学以及脂质双分子层体系中的磁取向。利用^<31>P核磁共振发现，在液晶-凝胶相变温度以上，蜂蜡- dmpc双层体系形成了长轴平行于磁场的磁性取向细长囊泡。在与脂质双分子层结合的^<13>C标记的蜂群蛋白上观察到^<13>C核磁共振谱。最后发现蜂毒蛋白采用平均轴平行于双层法线的跨膜α-螺旋结构。(2)合成了流感病毒HA2 (1-27) n端片段，并用固体核磁共振光谱技术对其进行了研究。结果发现，在酸性和碱性条件下，片段的n端部分形成α-螺旋，与膜的相互作用表现出不同的行为。(3)合成了流感病毒M2蛋白的跨膜片段（M2- tmp），并采用^<15>N固体核磁共振光谱对其进行了研究。结果表明，DOPC和DMPC双分子体系的α-螺旋轴分别偏离正常双分子层33°和37°。(4)采用中子散射法研究流感融合肽的膜插入行为。结果表明，螺旋轴与双分子层法线倾斜55°，螺旋位于双分子层表面附近。

In this research project, we have studied to understand the molecular mechanism of viral infectn using influenza fusion peptides. We have particularly focused to determine the structure and orientation of the peptides bund to the membrane. Following results were obtained in this research project.(1) 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 ^<31>P and ^<13>C NMR spectroscopy. Using ^<31>P 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. ^<13>C NMR spectra were observed on the ^<13>C labeled melittin bound to the lipid bilayers. Finally, it was found that melittin adopts a transmembrane α-helix whose average axis is parallel to the bilayer normal.(2) N-terminal fragment of HA2 (1-27) in influenza virus was synthesized and investigated by solid state NMR spectroscopy. It was found that the N-terminal part of the fragment forms α-helix and showed different behavior of interaction with membrane at acidic pH from that at basic pH condition.(3) Transmembrane fragment of M2 protein of influenza virus (M2-TMP) was synthesized and investigated by means of ^<15>N solid state NMR spectroscopy. The results indicate that the α-helical axis is tilting by 33° and 37° from the bilayer normalin the DOPC and DMPC bilayer systems, respectively.(4) Membran insertion behavior of influenza fusion peptide was investigated by a neutron scattering method. The results indicate that the helical axis is tilting by 55° from the bilayer normal and the helix is located around the surface of bilayers.