Solid-State NMR Study of Structure-Function Relationship of Protein Induced in the Signal Transduction Pathway at the Membrane Surface

2003年，我们建立了将~(13)C同位素标记引入PLC-δ1PH结构域的技术，并利用固体核磁共振技术对膜表面的~(13)H结构域进行了结构研究。2004年，(1)研究了在带负电的膜表面诱导的PH结构域的膜结合结构的变化，(2)建立了PLC-δ1剩余结构域(EF-Hand，XY和C2)的~&lt；13&gt；C同位素标记方法。在20%的酸性脂质磷脂酰丝氨酸存在下，PH结构域具有与溶液中相似的结构，而不是先前报道的在电中性膜表面观察到的膜结合结构。固体核磁共振谱的结构变化表明，带负电荷的膜表面与PH结构域的带电残基之间的静电斥力阻止了PH结构域的两亲性α2-螺旋与膜疏水内层之间的非特异性疏水相互作用，从而导致了PH结构域在中性膜表面独特的膜结合结构。在带负电荷的膜表面也观察到PH结构域的迁移率增加。PH结构域的动态结构对膜脂组成的依赖可能与PLC-δ1对局部脂质组成的生理变化的反应有关，如脂质微区的形成和脂质双层不对称性的改变。本研究建立了PH-EF片段、EF-Hand结构域和完整PLC-δ1的固体核磁共振标记和制备方法。这些样品将被用来研究膜表面PLC-δ1结构域之间的相互作用。

In 2003, we have established a technique of introduction of ^<13>C isotope labels into PLC-δ1 PH domain, and performed structural study of the PH domain at the membrane surface by using the solid state ^<13>C NMR spectroscopy. In 2004, (1)changes of the membrane binding structure of the PH domain induced at the negatively charged membrane surface were investigated, and (2)methods of ^<13>C isotope labeling of the remaining domains (EF-hand, XY, and C2) of PLC-δ1 were established.The structure of the PH domain was found to be strongly influenced by acidic lipid contents of the membranes. In the presence of 20% of acidic lipid, phosphatidylserine, PH domain takes a structure similar to that in the solution instead of the previously reported membrane-binding structure observed at the electrically neutral membrane surface. The structural changes observed by the solid-state ^<13>C NMR spectroscopy revealed that the electrostatic repulsions between the negatively charged membrane surface and the charged residues of the PH domain prevent non-specific hydrophobic interaction between the amphipathic α2-helix of the PH domain and the hydrophobic inner layer of the membrane which causes the unique membrane-binding structure of the PH domain at the neutral membrane surface. Increase in the mobility of the PH domain was also observed at the negatively charged membrane surface. Those dependences of the dynamic structure of the PH domain on the lipid composition of membrane might related to responses of the PLC-δ1 to physiological changes of local lipid compositions such as a formation of lipid micro domain and a change of asymmetry of lipid bilayer.Methods of ^<13>C labeling and preparation of solid state NMR samples of the PH-EF fragment, EF-hand domain and intact PLC-δ1 have been established in this year. These samples will be utilized to investigate inter-domain interactions between PLC-δ1 domains at the membrane surface.