1 research outputs found
Scaling the Functional Nanopore (FuN) Screen: Systematic Evaluation of Self-Assembling Membrane Peptides and Extension with a K<sup>+</sup>‑Responsive Fluorescent Protein Sensor
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 analyzed 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 analyzed
with the FuN screen and to what extent alternative fluorescent protein
sensors can be adapted. Focusing 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. Further,
the FuN screen is extended to K+-specific fluorescent protein
sensors and now provides a capacity to assess the specificity of a
nanopore or ion channel. Finally, a comparison to high-resolution
biophysical and electrophysiological studies in planar lipid bilayers
provides an experimental benchmark for future studies