Molecular basis of membrane stability and dynamics

Dit proefschrift is onderverdeeld in vier verschillende delen, die overeenkomen met de vier verschillende onderwerpen die ik heb gewerkt . In hoofdstukken 2-4, bestudeerden we het werkingsmechanisme van antimicrobiële peptiden. We concentreerden ons op twee korte peptiden, de cyclische BPC194 en zijn lineaire analoge BPC193. Hoewel beide bezitten dezelfde aminozuursequentie, maar het cyclische peptide kan microbiële cellen te doden. Met behulp van een combinatie van fluorescentie-gebaseerde technieken met moleculaire dynamica simulaties, kwamen we erachter dat het cyclisch peptide plooien in de juiste conformatie op membraanbindende, inserts diep, vormt poriën en fuseren de membranen . In hoofdstuk 5, wij ontwerpen en gesynthetiseerd een DNA - peptide hybride te moduleren de oligomerisatie van een membraan kanaal met behulp van complementaire DNA-strengen of een G-quadruplex motief. In beide gevallen heeft de daaruit zender heeft voorkeur voor een specifieke oligomere toestand van een vaste grootte . In hoofdstuk 6 hebben we de effecten van koolhydraten op het membraan organisatie. We combineerden fluorescentie microscopie met moleculaire dynamische simulaties en ontdekte dat alleen de niet-reducerende koolhydraten, sucrose en trehalose, hebben een effect op de lipid raft organisatie. Die twee disachariden worden meestal gesynthetiseerd door verschillende organismen, om te overleven in extreme uitdroging omstandigheden. In hoofdstuk 7 bestudeerden we de lokalisatie, oligomerisatie en dynamiek van verschillende plasmamembraan aminozuur transporters van S. cerevisiae. De verdeling van deze eiwitten in het membraan niet homogeen. We kwamen erachter dat de reden dat de extreem trage verspreiding die we gemeten voor die eiwitten samen met hun lage bedragen zou kunnen zijn

Structural basis for the enhanced activity of cyclic antimicrobial peptides:The case of BPC194

AbstractWe report the molecular basis for the differences in activity of cyclic and linear antimicrobial peptides. We iteratively performed atomistic molecular dynamics simulations and biophysical measurements to probe the interaction of a cyclic antimicrobial peptide and its inactive linear analogue with model membranes. We establish that, relative to the linear peptide, the cyclic one binds stronger to negatively charged membranes. We show that only the cyclic peptide folds at the membrane interface and adopts a β-sheet structure characterised by two turns. Subsequently, the cyclic peptide penetrates deeper into the bilayer while the linear peptide remains essentially at the surface. Finally, based on our comparative study, we propose a model characterising the mode of action of cyclic antimicrobial peptides. The results provide a chemical rationale for enhanced activity in certain cyclic antimicrobial peptides and can be used as a guideline for design of novel antimicrobial peptides

Single liposome analysis of peptide translocation by the ABC transporter TAPL

ATP-binding cassette (ABC) transporters use ATP to drive solute transport across biological membranes. Members of this superfamily have crucial roles in cell physiology, and some of the transporters are linked to severe diseases. However, understanding of the transport mechanism, especially of human ABC exporters, is scarce. We reconstituted the human lysosomal polypeptide ABC transporter TAPL, expressed in Pichia pastoris, into lipid vesicles (liposomes) and performed explicit transport measurements. We analyzed solute transport at the single liposome level by monitoring the coincident fluorescence of solutes and proteoliposomes in the focal volume of a confocal microscope. We determined a turnover number of eight peptides per minute, which is two orders of magnitude higher than previously estimated from macroscopic measurements. Moreover, we show that TAPL translocates peptides against a large concentration gradient. Maximal filling is not limited by an electrochemical gradient but by trans-inhibition. Countertransport and reversibility studies demonstrate that peptide translocation is a strictly unidirectional process. Altogether, these data are included in a refined model of solute transport by ABC exporters

The Molecular Basis for Antimicrobial Activity of Pore-Forming Cyclic Peptides

The mechanism of action of antimicrobial peptides is, to our knowledge, still poorly understood. To probe the biophysical characteristics that confer activity, we present here a molecular-dynamics and biophysical study of a cyclic antimicrobial peptide and its inactive linear analog. In the simulations, the cyclic peptide caused large perturbations in the bilayer and cooperatively opened a disordered toroidal pore, 1–2 nm in diameter. Electrophysiology measurements confirm discrete poration events of comparable size. We also show that lysine residues aligning parallel to each other in the cyclic but not linear peptide are crucial for function. By employing dual-color fluorescence burst analysis, we show that both peptides are able to fuse/aggregate liposomes but only the cyclic peptide is able to porate them. The results provide detailed insight on the molecular basis of activity of cyclic antimicrobial peptide

Dual Action of BPC194: A Membrane Active Peptide Killing Bacterial Cells

Membrane active peptides can perturb the lipid bilayer in several ways, such as poration and fusion of the target cell membrane, and thereby efficiently kill bacterial cells. We probe here the mechanistic basis of membrane poration and fusion caused by membrane-active, antimicrobial peptides. We show that the cyclic antimicrobial peptide, BPC194, inhibits growth of Gram-negative bacteria and ruptures the outer and inner membrane at the onset of killing, suggesting that not just poration is taking place at the cell envelope. To simplify the system and to better understand the mechanism of action, we performed Förster resonance energy transfer and cryogenic transmission electron microscopy studies in model membranes and show that the BPC194 causes fusion of vesicles. The fusogenic action is accompanied by leakage as probed by dual-color fluorescence burst analysis at a single liposome level. Atomistic molecular dynamics simulations reveal how the peptides are able to simultaneously perturb the membrane towards porated and fused states. We show that the cyclic antimicrobial peptides trigger both fusion and pore formation and that such large membrane perturbations have a similar mechanistic basi