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

Sushil Pangeni,Jigneshkumar Dahyabhai Prajapati,Ulrich Kleinekathöfer,Mathias Winterhalter,Jayesh Bafna,Mohamed Nilam,Werner M Nau

doi:10.1002/anie.202016943

Sushil Pangeni, Jigneshkumar Dahyabhai Prajapati + Show 5 more

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https://doi.org/10.1002/anie.202016943

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Abstract

Quantifying the passage of the large peptide protamine (Ptm) across CymA, a passive channel for cyclodextrin uptake, is in the focus of this study. Using a reporter‐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. Furthermore, we reconstituted single CymA channels into 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, in agreement with the liposome study. Applied‐field and steered molecular dynamics simulations added an atomistic view of the permeation events. It can be concluded that a concentration gradient of 1 μm Ptm leads to a translocation rate of about one molecule per second and per channel.

Highlights

Rapid and label-free monitoring of drug uptake into Gram-negative bacteria is a bottleneck in antibiotic drug discovery
The outer membrane of Gram-negative bacteria acts as a selective barrier for the uptake of small molecules including antibiotics and antimicrobial peptides
Ensemble Measurements of Protamine Translocation through CymA by the Tandem Membrane Assay In a first series of experiments, we investigated the translocation of Ptm across the ∆CymA channel by using the previously introduced tandem membrane assay.[18,19,23]

Summary

Introduction

Rapid and label-free monitoring of drug uptake into Gram-negative bacteria is a bottleneck in antibiotic drug discovery. The outer membrane of Gram-negative bacteria acts as a selective barrier for the uptake of small molecules including antibiotics and antimicrobial peptides. Typical examples are cationic antimicrobial peptides that have attracted wider interest as substitutes for classical antibiotics.[8–16] Whether these polycationic peptides permeate using a self-promoted pathway or through channel proteins remains an open but crucial question in understanding their antimicrobial activity and other putative functions. One such molecule is protamine (Ptm) which is a 32 amino acid-long polycationic peptide with a molecular mass of 5.1 kDa that contains 21 arginine residues (Figure 1A). The mode of action, i.e., self-promoted uptake or uptake via a protein channel, is unknown

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