基于随机增量截断和真空紫外CD光谱的蛋白质二级结构预测
Secondary structure prediction of protein constructs using random incremental truncation and vacuum-ultraviolet CD spectroscopy
摘要：
在果蝇中发现了一种新的尿嘧啶-DNA降解蛋白因子（称为UDE），与其他尿嘧啶-DNA结合或加工因子没有显着的结构和功能同源性。确定的3D结构的UDE将是一个真正的突破，在描述的分子机制的作用的UDE催化，以及在一般尿嘧啶识别和核酸酶的行动。革命性的ESPRIT技术被应用于新的蛋白质UDE，以克服在缺乏结构域内容和排列的精确信息的情况下鉴定可溶性表达构建体的问题。从创建的众多截断的UDE结构中选择了九个样本来解嵌结构和功能关系。用神经网络进行VUVCD，以确定UDE及其截短变体的二级结构含量和位置。定量分析证明了全长蛋白质的排他性α-螺旋含量，其保留在截短的构建体中。与截短的蛋白质片段相比，α-螺旋捆绑的分区表示新的结构域边界，其不同于通过基于序列的比对确定的化蛹昆虫中的UDE同源物的保守基序。在这里，我们证明了ESPRIT和VUVCD与NN的组合提供了显着的结构描述的UDE，并导致有用的截断构建进一步详细的功能研究。
英文摘要：
A novel uracil-DNA degrading protein factor (termed UDE) was identified in Drosophila melanogaster with no significant structural and functional homology to other uracil-DNA binding or processing factors. Determination of the 3D structure of UDE will be a true breakthrough in description of the molecular mechanism of action of UDE catalysis, as well as in general uracil-recognition and nuclease action. The revolutionary ESPRIT technology was applied to the novel protein UDE to overcome problems in identifying soluble expressing constructs given the absence of precise information on domain content and arrangement. Nine specimen from the created numerous truncated constructs of UDE were choosen to dechiper structural and functional relationships. VUVCD with neural network was performed to define the secondary structure content and location of UDE and its truncated variants. The quantitative analysis demonstrated exclusive α-helical content for the full-length protein, which is preserved in the truncated constructs. Partition of α-helical boundles comparison with the truncated protein segments denoted new domain boundaries which differ from the conserved motifs determined by sequence-based alignment of UDE homologues in pupating insects. Here we demonstrate that combination of ESPRIT and VUVCD with NN provided significant structural description of UDE and resulted useful truncated constructs for further detailed functional studies.
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