Characterisation of a Novel Molecular Tension Probe to Investigate Translocation of Colicin E9 across the Outer Membrane

Previous studies have hinted at the presence of a mechanical force that unfolds Colicin E9 (ColE9) at the cell surface, enabling it to be translocated into a target bacterial cell. This hypothesis has yet to be proven, though the Tol system is speculated to be the origin of this force. Numerous Förster resonance energy transfer (FRET) studies have been carried out to elucidate the mechanism of colicin translocation, a protein with implications for medicine. However, none of these single-molecule studies have been able to directly probe mechanical unfolding of ColE9 at the cell surface. Here, a novel molecular tension probe (MTP) has been proposed, utilising a dsDNA duplex, labelled with a FRET acceptor-donor fluorophore pair, inserted between structural domains of ColE9. The force required to unzip the dsDNA duplex (10-15pN) has been measured by force spectroscopy to be sufficient to unfold colicin (Cocco, Monasson and Marko, 2002; Robinson, 2013; Vankemmelbeke et al., 2013). Should an unfolding force be present, the MTP would observe it as a decrease in FRET efficiency. The proposed MTP model requires the synthesis of two ssDNA-protein chimeras. Described here are the results of purification and modification protocols, designed to engineer ColE9 domains into suitable components for the MTP. Progress has been made towards the generation of a final MTP, with additional modifications and revisions to the thiol coupling strategy planned. Also described is a structural bioinformatics project exploring readily available protein data bank (PDB) database files belonging to the OMP families OmpF, BtuB and VDAC. Symmetry analysis of these families has discovered preferred patches of intermolecular interaction on the outer surface of their β-barrels. Investigations into these patches have suggested they may be conserved through evolution across these divergent OMP families, first arising in a distant common ancestor.