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<13>在PLC-δ1 PH结构域中引入~（13）C同位素标记的技术，并利用固体~（13）C NMR谱对膜表面的PH结构域进行了结构研究<13>。2004年，（1）研究了在带负电荷的膜表面诱导的PH结构域的膜结合结构的变化，（2）建立了<13>PLC-δ1的剩余结构域（EF-手、XY和C2）的^ C同位素标记方法，发现PH结构域的结构强烈地受膜的酸性脂质含量的影响。在20%的酸性脂质，磷脂酰丝氨酸，PH域的存在下，采取类似的结构，在溶液中，而不是以前报道的膜结合结构观察到的电中性膜表面。固态13 C NMR谱观察到的结构变化<13>表明，带负电荷的膜表面与PH结构域的带电荷残基之间的静电排斥阻止了PH结构域的两亲性α2-螺旋与膜的疏水内层之间的非特异性疏水相互作用，这导致PH结构域在中性膜表面的独特膜结合结构。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质组成的依赖性可能与PLC-δ1对局部脂质组成的生理变化（如脂质微结构域的形成和脂双层不对称性的改变）的反应有关<13>。这些样品将用于研究膜表面PLC-δ1结构域之间的结构域间相互作用。

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.