Folding and assembly of de novo designed transmembrane β-barrels

The outer membrane poses a formidable permeability barrier against the treatment of Gram-negative bacterial infection, and, constituting a vital element implicated in its maintenance, are integral outer membrane proteins (OMPs). As these proteins all form a ubiquitous transmembrane β-barrel subunit, OMPs present an opportune antimicrobial target. Many unanswered questions regarding OMP biogenesis remain to be addressed. Recent computational design of de novo transmembrane β-barrels (TMBs) offers an opportunity to study the sequence features required for OMP folding, leveraging their small size and lack of evolutionary history. Preliminary characterisation of two de novo TMBs; TMB2.3 and TMB2.17, has demonstrated their insertion into liposomes. This thesis continues this work, performing a comprehensive evaluation of their folding in vitro, before investigation of their behaviour in an in vivo context. Chapter 3 explores TMB stability within lipid bilayers of differing aliphatic chain length, and deeper investigation into the folding of TMB2.17 reveals that its aggregation propensity is a key modulator of folding ability, which may be adjusted through substitution of key structural features. Chapter 4 considers TMB biogenesis in vivo, demonstrating that both TMBs can fold into the E. coli outer membrane, albeit inefficiently, when appended to a suitable signal sequence, thus, the sole functionality to form a β-barrel structure is sufficient for their targeting from the periplasm and insertion into the bacterial OM. Further in vitro investigations into chaperone-TMB interactions revealed that whilst cytoplasmic chaperones exhibit similar binding patterns to TMB peptides, periplasmic chaperones recognise distinct interaction regions. Furthermore, TMB2.3, but not TMB2.17, displays dependence on BAM for its insertion into lipid bilayers in vitro, implying varied mechanisms of outer membrane insertion in vivo. Overall, this thesis demonstrates that de novo designed TMBs constitute a valuable tool to further inform OMP biogenesis, highlighting key sequence features implicated in OMP folding and biogenesis.