Small, hydrophilic molecules, including most important antibiotics in clinical use, cross the Gram-negative outer membrane through the water-filled channels provided by porins. We have determined the X-ray crystal structures of the principal general porins from three species of Enterobacteriaceae, namely Enterobacter aerogenes, Enterobacter cloacae, and Klebsiella pneumoniae, and determined their antibiotic permeabilities as well as those of the orthologues from Escherichia coli. Starting from the structure of the porins and molecules, we propose a physical mechanism underlying transport and condense it in a computationally efficient scoring function. The scoring function shows good agreement with in vitro penetration data and will enable the screening of virtual databases to identify molecules with optimal permeability through porins and help to guide the optimization of antibiotics with poor permeation.

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Biological channels facilitate the exchange of molecules across membranes,b ut general tools to quantify transport are missing. Electrophysiology is the method of choice to study the functional properties of channels.However, analyzing the current fluctuation of channelstypically does not identify successful transport, that is,d istinguishing translocation from binding.T odistinguish both processes,weadded an additional barrier at the channel exit acting as am olecular counter.T oi dentify permeation, we compare the molecule residence time in the native channel with one that is chemically modified at the exit. We use the well-studied outer membrane channel from E. coli, OmpF.P osition 181, whichi sb elowt he constriction region, was subsequently mutated into cysteine (E181C) in an otherwise cysteine-free system, then functionalized by covalent binding with one of the two blockers MTSES or GLT. We measured the passage of model peptides, mono-, tri-, hepta-arginine and of norfloxacin, as an example for antibiotic permeation.