Secondary Structure Prediction of Protein Constructs Using Random Incremental Truncation and Vacuum-Ultraviolet CD Spectroscopy

Pukáncsik, Mária and Orbán, Ágnes and Nagy, Kinga and Matsuo, Koichi and Gekko, Kunihiko and Kézsmárki, István and Vértessy, Beáta (Grolmuszné) (2016) Secondary Structure Prediction of Protein Constructs Using Random Incremental Truncation and Vacuum-Ultraviolet CD Spectroscopy. PLOS ONE, 11 (6). e0156238. ISSN 1932-6203

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Abstract

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 is excepted to provide key information on the description of the molecular mechanism of action of UDE catalysis, as well as in general uracil-recognition and nuclease action. Towards this long-term aim, the random library 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 constructs of UDE were chosen to decipher structural and functional relationships. Vacuum ultraviolet circular dichroism (VUVCD) spectroscopy was performed to define the secondary structure content and location within UDE and its truncated variants. The quantitative analysis demonstrated exclusive alpha-helical content for the full-length protein, which is preserved in the truncated constructs. Arrangement of alpha-helical bundles within the truncated protein segments suggested new domain boundaries which differ from the conserved motifs determined by sequence-based alignment of UDE homologues. Here we demonstrate that the combination of ESPRIT and VUVCD spectroscopy provides a new structural description of UDE and confirms that the truncated constructs are useful for further detailed functional studies.

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