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.

在2003年，我们建立了一种将 ^<13> C同位素标记引入PLC-Δ1pH结构域的技术，并使用固态 ^<13> c NMR光谱法对膜表面的pH结构域进行了结构研究。在2004年，研究了（1）研究了在负电荷的膜表面诱导的pH结构域的膜结合结构的变化，（2）在PLC-δ1的其余结构域（EF手，XY和C2）的 ^<13> c同位素标记的方法中建立了pH结构域的结构。在存在20％的酸性脂质的磷脂酰丝氨酸的情况下，pH结构域采用与溶液相似的结构，而不是在电中心膜表面观察到的先前报道的膜结合结构。固态 ^<13> c NMR光谱观察到的结构变化表明，负电荷的膜表面与pH结构域的带电残留物之间的静电排斥阻止了pH域和膜的质量质量的中性层的隔离层之间的非特异性降水层之间的非特异性疏水相互作用，从而导致了膜的内部层面层，这会导致膜的质量频率，从而导致了膜的per embranation ph sufders fraphip。膜表面。在负电荷的膜表面也观察到pH结构域的迁移率的增加。 pH结构域动态结构对膜的脂质组成的依赖性可能与PLC-Δ1对局部脂质组成的物理变化的反应有关，例如脂质微型结构型的形成以及脂质双层的不对称变化，lipid bilayer的不对称性。 PLC-Δ1已于今年建立。这些样品将用于研究膜表面PLC-Δ1结构域之间的域间相互作用。