Nanopores comprise a versatile class of membrane proteins that carry out a range of key physiological functions and are increasingly developed for different biotechnological applications. Yet, a capacity to study and engineer protein nanopores by combinatorial means has so far been hampered by a lack of suitable assays that combine sufficient experimental resolution with throughput. Addressing this technological gap, the functional nanopore (FuN) screen now provides a quantitative and dynamic readout of nanopore assembly and function in the context of the inner membrane of Escherichia coli. The assay is based on genetically encoded fluorescent protein sensors that resolve the nanopore-dependent influx of Ca 2+ across the inner membrane of E. coli. Illustrating its versatile capacity, the FuN screen is first applied to dissect the molecular features that underlie the assembly and stability of nanopores formed by the S 21 68 holin. In a subsequent step, nanopores are engineered by recombining the transmembrane module of S 21 68 with different ring-shaped oligomeric protein structures that feature defined hexa-, hepta-, and octameric geometries. Library screening highlights substantial plasticity in the ability of the S 21 68 transmembrane module to oligomerize in alternative geometries, while the functional properties of the resultant nanopores can be fine-tuned through the identity of the connecting linkers. Overall, the FuN screen is anticipated to facilitate both fundamental studies and complex nanopore engineering endeavors with many potential applications in biomedicine, biotechnology, and synthetic biology.

The bacterial transpeptidase Sortase A (SrtA) is a surface enzyme of gram-positive pathogenic bacteria. It has been shown to be an essential virulence factor for the establishment of various bacterial...

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.