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Secondary structure prediction of protein constructs using random incremental truncation and vacuum-ultraviolet CD spectroscopy

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.