Biophysical Characterization of the Recombinant Importin-Alpha from Neurospora crassa
Biophysical Characterization of the Recombinant Importin-Alpha from Neurospora crassa
复制标题
粗糙脉孢菌重组导入蛋白-α 的生物物理表征
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发表时间：
2012
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通讯作者：
Takeda
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作者：
Takeda
粗糙脉孢菌已被广泛用作模式生物，并通过允许鉴定负责基因调控的许多代谢途径和机制而促进生物化学和分子生物学的发展。转录因子等核蛋白在细胞质中合成，需要转运到细胞核中发挥功能。经典的核输入途径依赖于由importin-α和importin-β组成的异源二聚体。为了获得N. crassa的IMPα基因的克隆、表达、纯化和结构研究。crassa（IMPα-Nc）。动态光散射实验和分析性尺寸排阻色谱表明，IMPα-Nc在不存在其自身抑制结构域或核定位序列（NLS）的情况下可以形成聚集体，但它主要是单体。此外，分子动力学模拟表明，与IMPα-Nc结合状态相比，apo单体IMPα-Nc在溶液中的稳定性可能较低。圆二色性分析表明IMPα-Nc的结构保守性高于哺乳动物蛋白。最后，IMPα-Nc模型的内凹表面具有高度的相似性，不同种属的IMPα与NLS的货物蛋白结合。然而，相对靠近NLS结合区的非保守氨基酸的存在可能影响IMPα-Nc与货物蛋白的结合特异性。
Neurospora crassa has been widely used as a model organism and contributed to the development of biochemistry and molecular biology by allowing the identification of many metabolic pathways and mechanisms responsible for gene regulation. Nuclear proteins such as transcription factors are synthesized in the cytoplasm and need to be translocated to the nucleus to exert their functions. The classical nuclear import pathway is dependent on the heterodimer composed of importin-α and importin-β. In an attempt to get structural information of the nuclear transport process in N. crassa, we present herein the cloning, expression, purification and structural studies with N-terminally truncated IMPα from N. crassa (IMPα-Nc). Dynamic light scattering experiments and analytical size exclusion chromatography indicated that the IMPα-Nc can form aggregates in the absence of its autoinhibitory domain or a nuclear localization sequence (NLS) however it is predominantly monomeric. Also molecular dynamics simulations suggest that apo monomeric IMPα-Nc may have low stability in solution compared to the IMPα-Nc bound state. In contrast, circular dichroism analysis revealed a high structural conservation of IMPα-Nc compared to mammalian protein. Finally, the IMPα-Nc model displayed high similarity of its inner concave surface, which binds the cargo proteins containing NLS, among IMPα from different species. However, the presence of non-conserved amino acids relatively close to the NLS binding region may influence the binding specificity of IMPα-Nc to cargo proteins.