Gertzen, C. | Search bioRxiv

bioRxiv2025

A novel biosensor for ferrous iron developed via CoBiSe:A computational method for rapid biosensor design

Genetically encoded biosensors enable monitoring of metabolite dynamics in living organisms. We present CoBiSe, a computational approach using Constraint Network Analysis to identify optimal insertion sites for reporter modules in molecular recognition elements (MREs). Applied to the iron-binding protein DtxR from Corynebacterium glutamicum, CoBiSe identified a flexible connective loop (residues 138-150) for inserting the reporter module, resulting in IronSenseR, a novel ratiometric biosensor for ferrous iron (Fe{superscript 2}). IronSenseR demonstrates high specificity for Fe{superscript 2} with dissociation constants of 1.55 {+/-} 0.08 {micro}M (FeSO4) and 2.44 {+/-} 0.28 {micro}M (FeCl2), while showing no binding to Fe3 and other divalent cations. In vivo assessment in Escherichia coli, Pseudomonas putida and Corynebacterium glutamicum confirmed IronSenseRs capability to detect changes in the intracellular iron pool. The creation of IronSenseR underlines that, by reducing search space and eliminating labor-intensive screening, CoBiSe streamlines biosensor development and enables precise creation of next-generation biosensors for diverse metabolites.

bioengineering↗

bioRxiv2021

A new twist in ABC transporter mediated multidrug resistance - Pdr5 is a drug/proton co-transporter

The two major efflux pump systems are involved in multidrug resistance (MDR): (i) ATP binding cassette (ABC) transporters and (ii) secondary transporters. While the former use binding and hydrolysis of ATP to facilitate export of cytotoxic compounds, the latter utilize electrochemical gradients to expel their substrates. Pdr5 from Saccharomyces cerevisiae is a prominent member of eukaryotic ABC transporters that are involved in MDR and used as a frequently studied model system. Although investigated for decades, the underlying molecular mechanisms of transport and specificity remain elusive. Here, we provide electrophysiological data on reconstituted Pdr5 demonstrating that this MDR efflux pump does not only actively translocate its substrates across the lipid bilayer, but generates a proton motif force in the presence of Mg2+-ATP and substrates by acting as a proton/drug co-transporter. Importantly, a strictly substrate dependent co-transport of protons was also observed in in vitro transport studies using Pdr5-enriched plasma membranes. Similar observations have not yet been reported for any other MDR efflux pump. We conclude from these results that the mechanism of MDR conferred by Pdr5 and likely other transporters is more complex than the sole extrusion of cytotoxic compounds and involves secondary coupled processes suitable to increase the effectiveness.

biochemistry↗