The influence of immobilisation and peptide structure on the bioactivity of model antimicrobial peptides

Lourens, Gertrude Dorothy (1999-11)

Thesis (M.Sc.) -- University of Stellenbosch, 1999.

Thesis

ENGLISH SUMMARY: Antimicrobial peptides play an integral role in the innate immunity of many plants and animals. An essential aspect of the activity of linear antimicrobial peptides is that they are cationic and have the ability to form an amphipathic a-helical secondary structure. Blondelle et al. identified cationic amphipathic peptides with pronounced antimicrobial activity in combinatorial libraries of model 18-residue peptides consisting mostly of Leu and Lys. One of these peptides (YKLLKLLLPKLKGLLFKL-NH2) and three N-terminal deletion analogues (16-, 14- and 12-residue peptides) were investigated in a structure-function study. Four peptide amides were synthesised using the Fmoc-polyamide solid phase peptide synthesis protocol. These four peptides were also prepared in immobilised form by an irreversible coupling of their carboxy-terminal amino acid to a (Gly)3 linker on an ethylenediamine-modified polydimethyl-acrylamide resin. The three longer peptide amides (NLK peptides) were purified using gel permeation chromatography and the shortest peptide by high performance chromatography (HPLC). The high chemical purity of the free peptide amides were confirmed by electrospray ionisation mass spectrometry (ES-MS) and analytical HPLC, while the integrity of the immobilised peptides were confirmed by amino acid analysis and protein sequencing. In the structure-function study the influence of peptide length, charge and amphipathicity on activity against Micrococcus luleus, Escherichia coli and red blood cells was evaluated. The influence of immobilisation on activity was tested against M luteus. The three longer free peptide amides had substantial antibacterial activity, but were more active towards the gram-positive bacterium, M luteus, than towards the gram-negative bacterium, E. coli. This specificity could be attributed to the difference in membrane structure of these two organisms, with the E. coli membrane differing because of its lipopolysaccharide layer. Interestingly, the activity against M luleus was in the same range than against the red blood cells. The haemolytic activity of these model peptides thus someehow correlated with their activity against gram-positive organisms. Similar activities were observed for 18-residue and the 16- residue peptides when tested against the same organism. However, the 16-residue peptide was more than fivefold less active against E. coli than M lufeus, while the other two peptides were about two fold less active. The difference in activity is probably because the 16-residue peptide having one less positive charge than the 18-residue peptide. The 14-residue peptide was less active than the two longer peptides, which could be the result of its smaller hydrophobic face it has when compared to the longer peptides. The 12-residue peptide amide tended to aggregate and was inactive. The reduced peptide length of the 12-residue peptide, its aggregation and its smaller hydrophobic face therefore influenced the peptide's activity. Some surface activity was, however, observed for the immobilised 12-residue peptide, probably because immobilisation prevented aggregation. Similar trends in activity against M luteus were observed when comparing the different peptide amides with their immobilised analogues, although the immobilised peptides were about 1000 fold less active. In conclusion, the amphipathic character, with particular emphasis on the size and density of the hydrophobic face, must be maintained to preserve antibacterial activity. Furthermore, immobilisation restricted the membrane activity of these peptides to only surface interaction, thus limiting any other interaction essential for activity. peptide having one less positive charge than the 18-residue peptide. The 14-residue peptide was less active than the two longer peptides, which could be the result of its smaller hydrophobic face it has when compared to the longer peptides. The 12-residue peptide amide tended to aggregate and was inactive. The reduced peptide length of the 12-residue peptide, its aggregation and its smaller hydrophobic face therefore influenced the peptide's activity. Some surface activity was, however, observed for the immobilised 12-residue peptide, probably because immobilisation prevented aggregation. Similar trends in activity against M luteus were observed when comparing the different peptide amides with their immobilised analogues, although the immobilised peptides were about 1000 fold less active. In conclusion, the amphipathic character, with particular emphasis on the size and density of the hydrophobic face, must be maintained to preserve antibacterial activity. Furthermore, immobilisation restricted the membrane activity of these peptides to only surface interaction, thus limiting any other interaction essential for activity.

AFRIKAANSE OPSOMMING: Antimikrobiese peptiede speel 'n integrale rol in die aangebore immuniteit van verskeie plante en diere. 'n Baie belangrike eienskap vir aktiwiteit van lineere antimikrobiese peptiede is dat hulle kationiese moet wees en 'n amfipatiese a-helikse sekondere struktuur moet aanneem. Blondelle et al. het kationiese amfipatiese peptiede met verhoogde antimikrobiese aktiwiteit, vanuit kombinatoriese biblioteke van model 18-residu peptiede wat meestal uit Leu en Lys bestaan, geYdentifiseer. Een van hierdie peptiede (YKLLKLLLPKLKGLLFKL-NH2) en drie N-terminaal delesie analoe (16-, 14- en 12-residu peptiede) is in 'n struktuur-funksie verwantskap ondersoek. Die vier peptiede is as peptiedamiede gesintetiseer deur gebruik te maak van die Fmoc-poliamied soliede-fase peptiedsintese protokol. Die vier peptiede is ook voorberei in die geYmmobiliseerde vorm deur hul karboksie-terminale residu onornkeerbaar te koppel aan 'n (Gly)3 verlengingsarm op 'n etileendiamien-gemodifiseerde polidimetielakrielamied hars. Die drie langer peptiedamiede (NLK-peptiede) is deur middel van gelpermeasie c1u-omatografie gesUlwer en die kortste peptied deur middel van hoe doeltreffendheid chromatografie (HPLC). Die hoe chemiese suiwerheid van die peptiedamiede is deur elektrosproei-ionisasie massaspektrometrie (ES-MS) en analitiese HPLC bevestig, terwl die integriteit van die geYmmobiliseerde peptiede deur aminosuur analise en proteien volgorde bepaling bevestig is. In die struktuur-funksie studie, is die invloed van peptiedlengte, lading en amfipatisiteit op die aktiwiteit teen micrococcus luteus, Escherichia coli en rooibloed selle ondersoek. Die invloed van immobilisering op antimikrobiese aktiwiteit is teen M. luteus ondersoek. Die drie langer peptiedamiede het beduidende aktiwiteit getoon, maar was meer aktief teen die gram-positiewe M. luteus as teen die gram-negatiewe E. coli. Die spesifisiteit is moontlik as gevolg van die verskille in membraanstruktuur tussen die twee organismes, met E. coli wat 'n lipopolisakkaried laag het. Wat tog interessant is, is dat die hemolitiese aktiwiteit van hierdie model peptiede ooreenkom met hul aktiwiteit teen M. luteus. Die 18-residu peptied en die 16- residue peptied het soortgelyke bioaktiwiteit teen dieselfde teikenselle getoon. Die 16-residu peptied is egter vyf maal minder aktief teen M. luteus as teen E. coli, terwyl die ander peptiede net twee maal minder aktief was. Die verskil in aktiwiteit kan moontlik is moontlik as gevolg van die een positiewe lading wat die 16-residu peptied minder het as die 18-residupeptied. Die 14-residu peptied het laer aktiwiteit getoon as die langer peptiede, wat moontlik toegeskryf kan word aan die kleiner hidrofobiese kant. Die 12-residu peptiedamied was geneig om te aggregeer en het geen aktiwiteit getoon nie. Die verkorte peptiedlengte, aggregasie-tendens en die kleiner hidrofobiese kant het dus hierdie peptied se aktiwiteit beinvloed. Oppervlakaktiwiteit is wel waargeneem vir die ge"immobiliseerde 12-residu peptied, moontlik omdat immobilisering die peptied verhoed om te aggregeer. Soortgelyke tendense in aktiwiteit teen M. luteus is waargeneem as die peptiedamiede se aktiwiteit met die van hul ge"immobiliseerde analoe vergelyk word. Die geimmobiliseerde peptiede is egter omtrent 'n 1000 maal minder aktief. Dit is dus belangrik, dat die amfipatiese karakter, spesifiek die grootte en digtheid van die hidrofobiese kant, behou moet word om antimikrobise aktiwiteit te bewaar. Verder het die immobilisering van die peptiede hulle tot oppervlakaktiwiteit beperk, wat dus enige ander interaksie wat nodig is vir aktiwiteit verklein.

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