Aminoglycosides are important clinical antibiotics but their molecular uptake mechanism is still not completely understood. Here we quantify and compare the passive transport of three aminoglycosides (kanamycin, gentamicin, and amikacin) across general or sugar specific porins of Escherichia coli (OmpF, OmpC, LamB and ChiP). Our analysis revealed that permeation of aminoglycosides (Kanamycin/Gentamycin/Amikacin) is about the same through ChiP (≈5/3/2 molecules/s), OmpF (≈10/15/<1 molecules/s) and OmpC (≈11/8/<1 molecules/s). In contrast, LamB of smaller pore diameter has no significant permeation (≤1/1/1 molecules/s, all values recalculated for a gradient of 10 uM). Biological assays confirmed the relevance of these translocations for antibiotic potency.
Characterizing protein-protein interaction on a single molecular level is a challenge, experimentally as well as the interpretation of the data. For example, Gram-negative bacteria contain protein complexes spanning the outer and inner cell wall being able to efflux effectively cell toxic substances. Within the search for antibiotics with novel modes of action, inhibition of the efflux pump assembly is an interesting possible target. Recent seminal work revealed the high-resolution structure of the tripartite composition TolC-AcrA-AcrB. Here, we reconstitute a single TolC homotrimer into a planar lipid membrane and follow the assembly of AcrA using the modulation of the ion current through TolC during binding. In particular, the presence of AcrA increases the average ionic current through TolC and, moreover, reduces the ion-current fluctuations caused by flickering of TolC. Here, we demonstrate that statistical properties of ion-current fluctuations (the power spectral density) provide a complementary measure of the interaction of the TolC-AcrA complex with potential inhibitors. Both characteristics, the average current and the current noise, taken into consideration together, provide more robust information.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.