The functional analysis of protein nanopores is typically conducted in planar lipid bilayers or liposomes exploiting high-resolution but low-throughput electrical and optical read-outs. Yet, the reconstitution of protein nanopores in vitro still constitutes an empiric and low-throughput process. Addressing these limitations, nanopores can now be analysed using the functional nanopore (FuN) screen exploiting genetically-encoded fluorescent protein sensors that resolve distinct nanopore-dependent Ca2+ in- and efflux patterns across the inner membrane of Escherichia coli. With a primary proof-of-concept established for the S2168, and thereof based recombinant nanopore assemblies, the question arises to what extent alternative nanopores can be analysed with the FuN screen. Focussing on self-assembling membrane peptides, three sets of 13 different nanopores are assessed for their capacity to form nanopores in the context of the FuN screen. Nanopores tested comprise both natural and computationally-designed nanopores while a comparison with high-resolution biophysical studies provides an experimental benchmark for future studies.
Nanopores constitute an important class of biotechnologically relevant proteins. Unlike binders and enzymes, their experimental characterization is limited to high-resolution, yet low throughput biophysical methods. Addressing this technological gap, the functional nanopore (FuN) screen now provides a versatile assay to study and engineer nanopores in Escherichia coli combining quantitative resolution with the ease, scalability, and throughput of cellular assays.
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