Quantitative assessment of porin-mediated solute transport in biomimetic membranes

Porins govern nutrient uptake and antibiotic influx in Gram-negative bacteria, making their characterization critical for understanding permeability, resistance mechanisms, and structure-function relationships. From a biotechnological point of view, they are effective tools for modulating the transport of substances across the outer bacterial membrane or for building catalytically active nanoreactors and biosensors. Quantitative data on mass transport through membranes is of great interest, but not trivial to obtain, as in vivo analyses are confounded by cellular complexity and variability. Here, we present a synthetic bottom-up approach, based on polymersomes containing reconstituted purified porins, enabling direct, quantitative measurement of substrate translocation, while minimizing interferences from native processes. Encapsulation of Gaussia luciferase allowed real-time monitoring of coelenterazine (CLZ) translocation across the polymeric membrane in the absence and presence of porins. The typically flash-type luciferase kinetics adapts a glow-type light emission profile, whose signal increases over time. This allows conclusions to be drawn about the substrate concentration accessible to the enzyme, enabling quantitative calculations of the transport rates. The novel approach was exemplarily used to compare the transport characteristics of three Escherichia coli porins: Outer membrane protein F (OmpF), a deletion variant selected for larger pore size OmpF∆, and Phosphoporin E (PhoE). OmpF∆ exhibited the highest transport rate of 78 molecules s^-1 per porin trimer, exceeding OmpF (10.8 molecules s^-1) more than sevenfold, whereas PhoE showed a lower rate of 2.8 molecules s^-1 for the neutral CLZ substrate. Analysis of two CLZ derivatives of slightly higher molecular mass and notably greater hydrophobicity revealed that transport through OmpF and OmpF∆ was reduced by half, whereas PhoE exhibited lower selectivity for the selected substrates. KEY POINTS: • Synthetic polymersomes enable direct, quantitative analysis of porin transport • OmpF∆ exhibits a sevenfold higher molecular flux than wildtype OmpF porins • The assay provides a versatile platform to study porin selectivity and permeability.