5 research outputs found
Applying flow cytometry to identify the modes of action of membrane-active peptides in a label-free and high-throughput fashion
Quantitative live-cell imaging of lipidated peptide transport through an epithelial cell layer
Cyclodextrin-Scaffolded Alamethicin with Remarkably Efficient Membrane Permeabilizing Properties and Membrane Current Conductance
Bacterial resistance to classical antibiotics is a serious
medical
problem, which continues to grow. Small antimicrobial peptides represent
a potential solution and are increasingly being developed as novel
therapeutic agents. Many of these peptides owe their antibacterial
activity to the formation of trans-membrane ion-channels resulting
in cell lysis. However, to further develop the field of peptide antibiotics,
a thorough understanding of their mechanism of action is needed. Alamethicin
belongs to a class of peptides called peptaibols and represents one
of these antimicrobial peptides. To examine the dynamics of assembly
and to facilitate a thorough structural evaluation of the alamethicin
ion-channels, we have applied click chemistry for the synthesis of
templated alamethicin multimers covalently attached to cyclodextrin-scaffolds.
Using oriented circular dichroism, calcein release assays, and single-channel
current measurements, the α-helices of the templated multimers
were demonstrated to insert into lipid bilayers forming highly efficient
and remarkably stable ion-channels
Tuning the double lipidation of salmon calcitonin to introduce a pore-like membrane translocation mechanism
A widespread strategy to increase the transport of therapeutic peptides across cellular membranes has been to attach lipid moieties to the peptide backbone (lipidation) to enhance their intrinsic membrane interaction. Efforts in vitro and in vivo investigating the correlation between lipidation characteristics and peptide membrane translocation efficiency have traditionally relied on end-point read-out assays and trial-and-error-based optimization strategies. Consequently, the molecular details of how therapeutic peptide lipidation affects it's membrane permeation and translocation mechanisms remain unresolved. Here we employed salmon calcitonin as a model therapeutic peptide and synthesized nine double lipidated analogs with varying lipid chain lengths. We used single giant unilamellar vesicle (GUV) calcein influx time-lapse fluorescence microscopy to determine how tuning the lipidation length can lead to an All-or-None GUV filling mechanism, indicative of a peptide mediated pore formation. Finally, we used a GUVs-containing-inner-GUVs assay to demonstrate that only peptide analogs capable of inducing pore formation show efficient membrane translocation. Our data provided the first mechanistic details on how therapeutic peptide lipidation affects their membrane perturbation mechanism and demonstrated that fine-tuning lipidation parameters could induce an intrinsic pore-forming capability. These insights and the microscopy based workflow introduced for investigating structure–function relations could be pivotal for optimizing future peptide design strategies.</p