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

Mária Pukáncsik,Darren Hart,István Kézsmárki,Damien Maurin,Ágnes Orbán,Kinga Nagy,Koichi Matsuo,Beata G Vertessy,Kunihiko Gekko

doi:10.1371/journal.pone.0156238

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 α-helical content for the full-length protein, which is preserved in the truncated constructs. Arrangement of α-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.

Highlights

Detailed knowledge of protein three-dimensional structure is indispensable for understanding the mechanism of protein action
Expression of soluble proteins using a library approach based upon random incremental truncation (ESPRIT) was used to create a comprehensive oversampled unidirectional truncation library from a fulllength ude gene [2,3,4,5,13] from which soluble variants were identified by screening
The six uracilDNA degrading factor (UDE) inserts were cloned into the plasmid vector pESPRIT002 that introduces restriction sites with exonuclease III sensitive and resistant overhangs at the termini to be truncated [32] and encoding an N-terminal hexahistidine tag and a C-terminal biotin acceptor peptide (BAP) [17] used here as an indicator of soluble expression (Fig 1)

Summary

Introduction

Detailed knowledge of protein three-dimensional structure is indispensable for understanding the mechanism of protein action. Secondary Structure Prediction of Protein Constructs by ESPRIT and Vacuum-UV CD Spectroscopy multidimensional NMR are the techniques of choice. For a detailed 3D structural determination by multidimensional NMR, the size of the protein is an important factor, and proteins larger than 30 kDa pose serious difficulties preventing structural determination [1]. There is no such size limitation in macromolecular X-ray crystallography, in this case, the need for well-diffracting crystal specimens is still a major bottleneck, especially in unstructured or flexible sequences that lack clearly identified domains. Methods to predict and help the design of crystallizable protein constructs are highly required. Generation and investigation of such deletion constructs may be a first and important step towards characterization of full-length proteins

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Conclusion