钙离子调控微丝切割蛋白中A6亚基解折叠的单分子力谱研究  

Single molecule force spectroscopy study of calcium regulated mechanical unfolding of the A6 domain of adseverin

作　　者：李鹏飞 曹毅[1] 秦猛[1] 王炜[1] 

机构地区：[1]南京大学物理学院固体微结构国家实验室,南京201193

出　　处：《物理学报》2017年第19期210-216,共7页Acta Physica Sinica

基　　金：国家自然科学基金(批准号:21522402;11674153;11374148;11334004);国家重点基础研究发展计划(批准号:2013CB834100)资助的课题~~

摘　　要：在生命活动中,金属离子扮演了非常重要的角色.微丝切割蛋白(adseverin)需要钙离子的活化才能行使其切割肌动蛋白微丝的功能.本文通过基于原子力显微镜的单分子力谱研究了微丝切割蛋白C端末的A6亚基在结合钙离子前后的力学解折叠机理.实验结果显示:在未结合钙离子时,A6的解折叠表现为两态过程;在结合钙离子后A6力学稳定性显著提高;同时,钙离子的结合使得A6解折叠过程中出现稳定的中间态.通过对中间态的链长的分析,我们推测了中间态对应着A6的N端部分解折叠.而这一部分的解折叠可以使得掩藏在该结构后的A5亚基中肌动蛋白微丝结合位点暴露,从而促使微丝切割蛋白执行功能.我们的实验结果为理解微丝切割蛋白的工作原理提供了新的实验证据.Adseverin is a member of calcium-regulated gelsolin superfamily existing in secretory cells, which functions as an actin severing and capping protein. Adseverin is comprised of six independently folded domains（A1-A6）, sharing high sequence identity（60%） with that of gelsolin（G1-G6）. Calcium binding can convert both adserverin and gelsolin from a globular structure into a necklace structure and expose the actin binding sites. However, compared with gelsolin,adseverin lacks a C-terminal extension. Our previous single molecule force spectroscopy studies indicated that the Cterminal helix is critical to the force regulated calcium activation of gelsolin. It remains largely unexplored how the calcium binding to adseverin is regulated by force.Here, using atomic force microscopy based single molecule force spectroscopy, we demonstrate that the mechanical unfolding of the sixth domain of adseverin（A6） can be significantly affected by calcium binding. In order to identify the unfolding events of A6 unambiguously, we construct a hetero-polyprotein（GB1-A6）4, in which A6 is spliced alternatively with well-characterized protein domain GB1. Therefore, in the force-extension traces, GB1 unfolding events can serve as a fingerprint to identify the unfolding signature of A6.In the absence of calcium, the unfolding traces for（GB1-A6）4 show two distinct categories of events. The higher force events with unfolding forces of ~180 pN and contour length increments of ~18 nm correspond to the unfolding of GB1. The other category of events with lower unfolding forces of ~25 pN and contour length increments of ~35 nm are attributed to the mechanical unfolding of A6. The unfolding force for A6 is similar to that for the structural homological protein, G6.However, in the presence of calcium ion, the unfolding force of A6 is dramatically increased to ~45 pN, indicating that the structure of A6 can be mechanically stabilized by calcium ion-binding. Moreover, we observe a clear mechanical unfolding intermediate state fo

关 键 词：微丝切割蛋白 单分子力谱 解折叠 中间态 

分 类 号：Q51[生物学—生物化学]