Functional Nanopore Screen: A Versatile High-Throughput Assay to Study and Engineer Protein Nanopores in Escherichia...

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 Ca²⁺ 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 S²¹68 holin. In a subsequent step, nanopores are engineered by recombining the transmembrane module of S²¹68 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 S²¹68 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.