关键词: 粗糙脉孢菌, 丝切蛋白, 肌动蛋白, 高速共沉淀, 微丝解聚活性

Abstract: 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 five α-helices and six β-strands. Of them, four internal β-strands were antiparallel, and all the β-strands were surrounded by the five α-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.

Key words: Neurospora crassa, cofilin, actin, high-speed co-sedimentation, F-actin depolymerization properties