Structural Determination and Membrane Interaction of the Antimicrobial Peptide Crabrolin

Frederiksen, Vera

4. term

Nanotechnology, Master

2009

57

Crabrolin er et kort antimikrobielt peptid (AMP) med 13 aminosyrer, som findes i giften fra den europæiske gedehams (Vespa crabro). AMP’er kan dræbe bakterier, og nogle er også hæmolytiske (kan skade røde blodlegemer). Crabrolin er kationisk (positivt ladet) med sekvensen NH2-LATVIKRLILPLF. I dette projekt blev peptidets tredimensionelle struktur og orientering i dodecylphosphocholin (DPC) miceller bestemt. Derudover blev det undersøgt, hvordan crabrolin interagerer med fosfolipider, og om det kan perforere fosfolipid-vesikler. Crabrolin blev syntetiseret ved solid-phase peptide synthesis (SPPS) og renset med højtryksvæskekromatografi (HPLC). Struktur og orientering i DPC-miceller blev bestemt med 2D-NMR-spektroskopi. pH-stabiliteten blev undersøgt med cirkulær dikroisme (CD). Interaktioner med miceller af natriumdodecylsulfat (SDS), DPC og dihexanoyl-phosphatidylcholin (DHPC) blev undersøgt ved titrering, og effekten på peptidets struktur blev fulgt med CD. Evnen til at perforere vesikler blev målt som farvestoflækage fra calcein-fyldte vesikler (DOPG og DPPC) ved fluorescensspektroskopi. Interaktionen for en mutant, Crabrolin W9, med 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DOPG), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholin (EDOPC) og dipalmitoylphosphatidylcholin (DPPC) blev undersøgt via UV-fluorescens fra tryptofan. Hæmolytisk aktivitet blev målt med absorbansspektroskopi, og antibakteriel aktivitet mod E. coli blev vurderet ved optisk densitet ved 600 nm (OD600). Resultaterne viste, at crabrolin danner en alfa-helix, hvor de kationiske (positivt ladede) aminosyrer er placeret på den ene side. I DPC-miceller er de hydrofobe (vandafvisende) rester i kontakt med micellens hydrofobe kerne, mens de polare rester ligger ved overfladen. Titrering med SDS og DPC gav en tydelig alfa-helikal struktur, mens DHPC primært gav en uordnet “random coil”. Crabrolin medførte mere calcein-frigivelse fra DOPG-vesikler end fra DPPC, hvilket tyder på stærkere virkning på negativt ladede membraner. Mutanten W9 indsatte sig helst i DOPG-vesikler, men kunne også indsætte i EDOPC- og fosfatidylcholin (PC) vesikler. Den hæmolytiske aktivitet for crabrolin, Crabrolin W9 og amidiseret crabrolin lå mellem 0,5 og 3,5 %, hvor den amidiserede variant var højest. I E. coli-aktivitetstests var den antibakterielle aktivitet af amidiseret crabrolin højere end for det oprindelige peptid.

Crabrolin is a short antimicrobial peptide (AMP) with 13 amino acids found in the venom of the European hornet (Vespa crabro). AMPs can kill bacteria, and some are also hemolytic (they can damage red blood cells). Crabrolin is cationic (positively charged) with the sequence NH2-LATVIKRLILPLF. This project determined the peptide’s three-dimensional structure and orientation in dodecylphosphocholine (DPC) micelles. It also examined how crabrolin interacts with phospholipids and whether it can perforate phospholipid vesicles. Crabrolin was synthesized by solid-phase peptide synthesis (SPPS) and purified by high-performance liquid chromatography (HPLC). Its structure and orientation in DPC micelles were determined using 2D NMR spectroscopy. pH stability was assessed by circular dichroism (CD). Interactions with micelles of sodium dodecyl sulfate (SDS), DPC, and dihexanoyl phosphatidylcholine (DHPC) were studied by titration, and changes in peptide structure were tracked by CD. Membrane perforation was measured as dye leakage from calcein-loaded vesicles (DOPG and DPPC) using fluorescence spectroscopy. The interaction of a mutant, Crabrolin W9, with 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DOPG), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC), and dipalmitoylphosphatidylcholine (DPPC) was studied via UV fluorescence of the tryptophan residue. Hemolytic activity was measured by absorbance spectroscopy, and antibacterial activity against E. coli was evaluated by optical density at 600 nm (OD600). By 2D NMR, crabrolin adopts an alpha-helix with its cationic residues located on one side. In DPC micelles, hydrophobic (water-repelling) residues face the micelle’s hydrophobic core, while polar residues remain at the surface. Titrations with SDS and DPC promoted an alpha-helical structure, whereas DHPC led to a largely random coil. Crabrolin caused greater calcein release from DOPG than from DPPC vesicles, indicating stronger action on negatively charged membranes. The W9 mutant preferentially inserted into DOPG vesicles, but also inserted into EDOPC and phosphatidylcholine (PC) vesicles. Hemolytic activity for crabrolin, Crabrolin W9, and amidated crabrolin ranged from 0.5 to 3.5%, with the amidated variant highest. In E. coli assays, amidated crabrolin showed higher antibacterial activity than the original peptide.

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