The Functional Nanopore Screen: A Versatile High-throughput Assay to Study and Engineer Protein Nanopores inEscherichia coli

Abstract

Nanopores comprise a versatile class of membrane proteins that carry out a range of key physiological functions and are increasingly exploited in many biotechnological applications. Yet, a capacity to study and engineer nanopores in the context of live cells has so far been hampered by a lack of suitable assays that provide sufficient experimental resolution and throughput. Addressing this technological gap, a newly developed Functional Nanopore (FuN) Screen now provides a highly quantitative read-out of nanopore function in E. coli. The assay is based on genetically-encoded fluorescent protein (FP) sensors that resolve the nanopore-dependent influx of Ca2+ across the inner membrane of E. coli. The FuN Screen is subsequently applied to dissect the molecular features that underlie the formation of nanopores by the S2168 holin. This membrane peptide plays a critical role in the S21 bacteriophage life cycle as it assembles into defined nm-sized nanopores to initiate lysis of the host cell. Genetic mapping experiments complemented with high-resolution electrical recordings shedding detailed light on the molecular determinants that underlie the formation of S2168 nanopores in the inner membrane. Overall, the FuN Screen is anticipated to facilitate both fundamental studies of nanopore functions and the construction of nanopores with tailored properties and function in E. coli.

Scaling the Functional Nanopore (FuN) Screen: Systematic Evaluation of Self-Assembling Membrane Peptides and Extension with a K+-Responsive Fluorescent Protein Sensor

“…A key bottleneck concerns our ability to study and engineer nanopores by experimental means which frequently relies on tailored expression and reconstitution protocols in lipid bilayers in vitro [5][6][7] . Addressing these limitations, the functional nanopore (FuN) screen exploits Ca 2+ -responsive fluorescent protein (FP) sensors to resolve the functional properties of a nanopore according to distinct Ca 2+ in-and efflux patterns across the E. coli inner membrane 8 (Fig. 1A).…”