Poster Presentation The 43rd Lorne Conference on Protein Structure and Function 2018

Differences in aggregation behaviour between a recombinantly expressed synthetic DYW-type Pentatricopeptide Repeat protein and a native protein from Arabidopsis thaliana (#225)

Santana Royan 1 , Bernard Guttman 2 , Jason Schmidberger 1 , Charlie Bond 1 , Ian Small 2
  1. School of Molecular Sciences, The University of Western Australia, Crawley, WA, Australia
  2. ARC Centre of Excellence in Plant Energy Biology, The University of Western Australia, Crawley, WA, Australia

Pentatricopeptide repeat (PPR) proteins are eukaryotic RNA binding proteins that are especially prevalent in terrestrial plants. Members of the PPR family in plants can be classified into two distinct subfamilies, the P subfamily and the PLS subfamily. A subclass of PLS subfamily PPR proteins contain a novel C-terminal domain, named the DYW deaminase domain due to its conserved terminal aspartate (D), tyrosine (Y), tryptophan (W) triplet. DYW type PPR proteins are implicated as trans-factors in C to U RNA editing in plant chloroplasts and mitochondria, although structural and mechanistic information about this process is lacking. To address this, we have expressed and purified a recombinant Arabidopsis thaliana DYW-type protein, AEF3, as an N-terminal His6-Maltose Binding Protein (MBP) fusion construct, as well as a designer DYW-type PPR protein, Syn3PLS-DYW, as an N-terminal Thioredoxin (Trx)-His6 fusion construct. Comparisons between the two using Size Exclusion Chromatography (SEC) and SEC with Multi-Angle Light Scattering (MALS) detectors indicates that while AEF3 is an aggregate at a size of 107 Daltons, Syn3PLS-DYW is monomeric, and interacts with a supplementary editing factor, MORF9. Crystallisation trials and Small Angle X-ray Scattering experiments are underway to obtain structural information of Syn3PLS-DYW, as well as it’s RNA binding and editing mechanisms.