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, 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 across 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 provided an atomistic view on the permeation. It can be concluded that a concentration gradient of 1 M Ptm leads to a translocation rate of about 1 molecule per second and per channel.

How to Enter a Bacterium: Bacterial Porins and the Permeation of Antibiotics

“…This 13 Å wide pore is suggested to have a role in the transport of large oligosaccharides due to the presence of a patch of aromatic residues, leading from the extracellular side to the constriction region of the pore as well. At the same time, this pore can be seen as an ideal candidate for biosensing. − Another hollow channel is the nucleoside-specific Tsx channel from E. coli , consisting of a 12-stranded oval-shaped barrel, which has also been affiliated to albicidin uptake. − It has a cross-section area of 10–12 × 3–5 Å, and the presence of a greasy slide along the channel introduces two binding sites for nucleosides, e.g., thymidine and uracil. Similarly, the sialic-acid-specific ( N -acetylneuraminic acid) channel NanC from E. coli also forms a 12-stranded barrel having an average diameter of 6.6 Å .…”

“…The change in the fluorescence intensity was provided as proof for porin-mediated transport of protamine. More recently, this technique in combination with the electrophysiology experiments led to estimation of the protamine permeation rate through the ΔCymA channel, equivalent to 1–2 molecules/s …”

“…More recently, this technique in combination with the electrophysiology experiments led to estimation of the protamine permeation rate through the ΔCymA channel, equivalent to 1−2 molecules/s. 117 5.2.2. Single-Channel Electrophysiology Assays.…”

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][40][41][42].…”