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

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.

The noncoding RNAs SNORD50A and SNORD50B-mediated TRIM21-GMPS interaction promotes the growth of p53 wild-type breast cancers by degrading p53

“…Circular dichroism (CD) spectroscopy is generally used as a tool to study protein secondary structure [ 47 , 48 ]. In this study, we examined the conformation of TRIM21 and GMPS interacting with SNORD50A/B using CD spectroscopy.…”

Synchrotron-radiation vacuum-ultraviolet circular dichroism spectroscopy in structural biology: an overview

“…This protein consists of 355 amino acid residues, beyond the upper limit of NMR measurements, and it has not been successfully crystallized, probably due to the predicted high conformational freedom of several segments. The VUVCD measurements were performed down to 170 nm for full-length UDE and its nine truncated fragments covering the full-length region, which were constructed using a random-screening method [ 85 ]. The VUVCD-NN analysis predicted a large α-helix content (62%, 13 segments) and a small amount of β-strands (8%, 7 segments) for the full-length protein, with most of these segments being preserved in the truncated fragments.…”

“…The arrangement of the α-helix bundles within the truncated fragments suggested new domain boundaries that differ from the conserved motifs determined by sequencebased alignment of UDE homologs. This new structural description of UDE forms a basis for further detailed functional studies [ 85 ].…”