粗糙脉孢菌丝切蛋白的纯化及微丝解聚活性分析

Purification and characterization of F-actin depolymerization properties of cofilin from Neurospora crassa

为探讨粗糙脉孢菌丝切蛋白(NcCof)的生化功能,对其进行纯化和微丝解聚活性分析。序列比对结果表明,NcCof N端第4位丝氨酸(S4)高度保守,推测其是NcCof发挥活性的重要位点,三级结构预测结果表明,它含有5个α-螺旋和6个β-折叠,中间的4个β折叠片处于反平行状态,5个α-螺旋围绕在β折叠片周围,具有典型的丝切蛋白/肌动蛋白解聚因子家族结构特征。利用点突变、亲和层析纯化得到蛋白NcCof、NcCof(S4A)和NcCof(S4D),进一步通过高速共沉淀等技术对其微丝解聚活性进行研究,结果表明,NcCof具有经典的微丝解聚活性,并具有剂量效应,突变蛋白NcCof(S4A)和NcCof(S4D)也具有解聚微丝的能力,但是NcCof(S4D)的解聚活性明显减弱。结果为进一步探讨NcCof调控肌动蛋白动态特性的分子机制奠定基础。

In order to clarify biochemical properties of cofilin from Neurospora crassa(NcCof),purification and characterization of F-actin depolymerization properties of NcCof were performed in this study.Sequences alignment indicated that Ser4 in the N-terminus of NcCof was highly conserved and might be important for its F-actin severing activity.Three-dimensional structure analysis showed that NcCof was composed of fiveα-helices and sixβ-strands.Of them,four internalβ-strands were antiparallel,and all theβ-strands were surrounded by the fiveα-helices,which were consistent with the typical structure of cofilin/ADF family.Subsequently,the S4 of NcCof was performed site-directed mutagenesis and two proteins NcCof(S4A)and NcCof(S4D)were obtained after purification.Furthermore,high-speed co-sedimentation assay was used to determine and compare the F-actin depolymerizing activity.The result showed that the control(NcCof)demonstrated the intrinsic and concentration dependent F-actiin depolymerizing activity,while this activity of both NcCof(S4A)and NcCof(S4D)displayed a weakening trend,especially the F-actin depolymerizing activity of NcCof(S4D)decreased significantly.This research conformed that S4 of NcCof was the key site for the F-actin depolymerization in Neurospora crassa,which would be helpful in further exploring the mechanisms of regulating actin dynamics by NcCof.