Folding Determinants of Transmembrane β-Barrels Using Engineered OMP Chimeras

Transmembrane beta-barrel proteins (OMPs) are highly robust structures for engineering and development of nanopore channels, surface biosensors, and display libraries. Expanding the applications of designed OMPs requires the identification of elements essential for beta-barrel scaffold formation and stability. Here, we have designed chimeric 8-stranded OMPs composed of strand hybrids of Escherichia coli OmpX and Yersinia pestis Ail, and identified molecular motifs essential for beta-barrel scaffold formation. For the OmpX/Ail chimeras, we find that the central hairpin strands beta 4-beta 5 in tandem are vital for beta-barrel folding. We also show that the central hairpin can facilitate OMP assembly even when present as the N- or C-terminal strands. Further, the C-terminal beta-signal and strand length are important but neither sufficient nor mutually exclusive for beta-barrel assembly. Our results point to a nonstochastic model for assembly of chimeric beta-barrels in lipidic micelles. The assembly likely follows a predefined nucleation at the central hairpin only when presented in tandem, with some influence from its absolute position in the barrel. Our findings can lead to the design of engineered barrels that retain the OMP assembly elements necessary to attain well-folded, stable, yet malleable scaffolds, for bionanotechnology applications.