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

膜表面信号转导通路诱导蛋白质结构-功能关系的固态核磁共振研究

基本信息

批准号：
15570164
负责人：
TUZI Satoru
金额：
$ 1.6万
依托单位：
University of Hyogo (2004)Himeji Institute of Technology (2003)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

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.

2003年，我们建立了一种将^<13>C同位素标签引入PLC-δ1 PH域的技术，并利用固态^<13>C核磁共振波谱对膜表面的PH域进行了结构研究。2004年，(1)研究了在带负电荷的膜表面诱导PH结构域膜结合结构的变化，(2)建立了PLC-δ1剩余结构域（EF-hand、XY和C2）的^<13>C同位素标记方法。PH结构域的结构被发现受到细胞膜酸性脂质含量的强烈影响。当20%的酸性脂质磷脂酰丝氨酸存在时，PH结构域呈现与溶液中相似的结构，而不是之前报道的在电中性膜表面观察到的膜结合结构。固态^<13>C核磁共振波谱观察到的结构变化表明，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-螺旋与膜的疏水内层之间的非特异性疏水相互作用，从而导致PH结构域在中性膜表面具有独特的膜结合结构。在带负电荷的膜表面也观察到PH结构域迁移率的增加。PH结构域的动态结构对膜脂质组成的依赖性可能与PLC-δ1对局部脂质组成生理变化的响应有关，如脂质微结构域的形成和脂质双分子层不对称性的改变。今年建立了^<13>C标记和制备PH-EF片段、EF-hand结构域和完整PLC-δ1的固态NMR样品的方法。这些样品将用于研究膜表面PLC-δ1结构域间的相互作用。