• Vera Frederiksen
4. semester, Nanoteknologi, Kandidat (Kandidatuddannelse)
Crabrolin is a 13 residue antimicrobial peptide (AMP) that is found in the venom of the european hornet Vespa Crabro. This AMP possesses haemolytic as well as antibacterial activity. Its sequence NH2-LATVIKRLILPLF places it in the group of cationic AMPs. In this project the tertiary structure and orientation of Crabrolin within Dodecylphosphocholine (DPC) micelles was determined. In addition the ability of Crabrolin to interact with phospholipids and perforate phospholipid vesicles was studied. Crabrolin was synthesised by Solid Phase Peptide Synthesis (SPPS) and purified by High Performance Liquid Chromatography (HPLC). The tertiary structure of Crabrolin as well as its orientation within DPC micelles was determined by 2-D NMR spectroscopy. The pH stability of Crabrolin was examined using Circular Dicroism (CD) spectroscopy. The ability of Crabrolin to interact with phospholipids was examined by titration with sodium dodecyl sulphate (SDS), DPC, and dihexanoyl phosphatidylcholin (DHPC) micelles. The effect of the micelles on the structure of Crabrolin was followed by CD spectroscopy. In addition the ability of Crabrolin to perforate vesicles was studied by dye leakage experiments with calcein containing vesicles, where the increase in fluorescence yield of calcein is followed over time by fluorescence spectroscopy. The interaction of a Crabrolin mutant (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 by UV fluorescence of the tryptophan residue. The haemolytic activity of Crabrolin was measured by absorbance spectroscopy and the antimicrobial activity by measuring optical density at 600 nm (OD600). By 2-D NMR the structure of Crabrolin was found to be α-helical with the cationic residues situated at one site of the helix. This helix was found to be situated with its hydrophobic residues in contact with the hydrophobic core of DPC and the polar residues situated at the surface of the DPC micelle. The titration of Crabrolin with SDS and DPC micelles disclosed an α-helical structure, while the titration with DHPC showed only random coil structure. The addition of Crabrolin to calcein loaded DOPG and DPPC vesicles lead to larger calcein release from DOPG than from DPPC. The study with Crabrolin W9 showed that it preferentially inserted into DOPG vesicles, however it was also found to insert into EDOPG and PC vesicles. The haemolytic activity of Crabrolin and its derivative, Crabrolin W9, as well as amidated Crabrolin was found to be in the range of 0.5 to 3.5 %, where amidated Crabrolin displayed the highest haemolytic activity. By activity assays on E.coli the antibacterial activity of amidated Crabrolin was found to be higher than that of Crabrolin.
SprogEngelsk
Udgivelsesdato2009
Antal sider57
Udgivende institutionAAU
ID: 17741587