Systematic Evaluation of Self-Assembling Membrane Peptides to Form Nanopores in the context of the Functional Nanopore Screen

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 holin, 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.