Large‐Peptide Permeation Through a Membrane Channel: Understanding Protamine Translocation Through CymA from Klebsiella Oxytoca. Issue 15 (3rd March 2021)

• Record Type: Journal Article
• Authors: Pangeni, Sushil; Prajapati, Jigneshkumar Dahyabhai; Bafna, Jayesh; Nilam, Mohamed; Nau, Werner M.; Kleinekathöfer, Ulrich; Winterhalter, Mathias
• Is Part Of: Angewandte Chemie international edition. Volume 60:Issue 15(2021)
• Page Start: 8089
• Page End: 8094
• Publication Date: 2021-03-03
• Subjects: electrophysiology -- membrane translocation assay -- molecular dynamics simulations -- outer membrane porins -- protamine; Chemistry -- Periodicals; 540
• DOI: 10.1002/anie.202016943
• Languages: English
• ISSNs: 1433-7851

Abstract

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. Abstract : Surprisingly, large peptides (Protamine, 5.1 kDa) can permeate through bacterial outer membrane channels. The use of fluorescence, electrophysiology, and all‐atom modeling allows to quantify the flux. This approach can be transferred to related problems.