Large‐Peptide Permeation Through a Membrane Channel: Understanding Protamine Translocation Through CymA from Klebsiella Oxytoca**

Abstract

Quantifying the passage of the large peptide protamine (Ptm) across CymA, apassive channel for cyclodextrin uptake,isinthe focus of this study.Using areporter-pair-based fluorescence membrane assay we detected the entry of Ptm into liposomes containing CymA. The kinetics of the Ptm entry was independent of its concentration suggesting that the permeation through CymA is the rate-limiting factor.F urthermore,w e reconstituted single CymA channelsi nto planar lipid bilayers and recorded the ion current fluctuations in the presence of Ptm. To this end, we were able to resolve the voltage-dependent entry of single Ptm peptide molecules into the channel. Extrapolation to zero voltage revealed about 1-2 events per second and long dwell times,i na greement with the liposome study.A pplied-field and steered molecular dynamics simulations added an atomistic view of the permeation events.Itcan be concluded that ac oncentration gradient of 1 mm Ptm leads to at ranslocation rate of about one molecule per second and per channel.

Cephalosporin translocation across enterobacterial OmpF and OmpC channels, a filter across the outer membrane

“…We adopted the recently developed FARMA (fluorescent artificial receptor-based membrane assay) method, that can be used for direct time-resolved monitoring of direct as well as porin-assisted permeation of antibiotics across isolated porins (Supplementary Fig. 7a ) 43 , 44 , 50 . Since the method can be used for both low as well as high-molecular-weight analytes, it should be equally applicable for fluorescence-based monitoring of antibiotic uptake.…”

What have molecular simulations contributed to understanding of Gram-negative bacterial cell envelopes?

“…In general, the simulations have been able to provide details of antibiotic-protein interactions, hydration levels of protein channels, flexibility of internal protein cavities as well as other conformational details that are not available from static structures. Due to space limitations we are unable to describe these studies in detail here, but we refer the reader to a number of key recent papers and a review [39–42].…”

Atomistic Simulation of Molecules Interacting with Biological Nanopores: From Current Understanding to Future Directions

“…The structural diversity of AMPs and their mechanisms of action have made this class of antimicrobials intriguing and have opened a variety of possibilities in rational and computational design. , While the mechanism of transport of these peptides across the outer membrane of Gram-negative bacteria is less understood, transport through bacterial channels is one of the suggested routes. For example, a recent study investigating protamine transport through the CymA channel hints toward this possibility …”