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Opredelitev vpliva izbranih aminokislinskih zamenjav v holesterol-vezavni domeni perfringolizina O
Filipović, Marina (Author), Anderluh, Gregor (Mentor) More about this mentor... This link opens in a new window, Lenarčič, Brigita (Co-mentor)

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Abstract
Perfringolizin O (PFO) je pomemben virulenčni dejavnik bakterije Clostridium perfringens. Izloča se kot vodotopni monomer in se veže izključno na membrane, ki vsebujejo holesterol. Znano je, da se citolitični mehanizem PFO prične z vezavo njegove četrte domene (D4) na plazmalemo evkariontske celice, ter da začetna vezava poteka preko štirih zank na dnu te domene. Za prepoznavo in vezavo holesterola v membrani sta ključna dva aminokislinska ostanka in sicer T490-L491, ki sta v literaturi predlagana kot holesterol-vezavni motiv (angl. cholesterol recognition motif; CRM). Kljub temu točen mehanizem specifične prepoznave in začetne vezave membranskega holesterola še ni docela pojasnjen. Cilj raziskovalnega dela je bil ovrednotiti, kako izbrane aminokislinske zamenjave v zankah D4 proteina PFO vplivajo na njegovo vezavo na različne modelne membranske sisteme. Različice PFO ter posameznih D4 smo izrazili v bakterijskem ekspresijskem sistemu E. coli in jih uspešno očistili z Ni-NTA afinitetno kromatografijo ter kromatografijo z ločevanjem po velikosti. Vpliv mutacij na aktivnost izbranih različic PFO smo ovrednotili z merjenjem hemolitične aktivnosti, pri čemer smo aktivnost vsake različice primerjali z aktivnostjo divjega tipa. Ugotovili smo, da so različice z bolj hidrofobnimi aminokislinskimi ostanki v predlaganem CRM bolj hemolitično aktivne. Pri testih vezave na modelne membranske sisteme smo ugotovili, da se različice D4 z aminokislinskimi zamenjavami v predlaganem CRM vežejo na multilamelarne vezikle ter na orjaške unilamelarne vezikle s 50 mol. % holesterola. Dokazali smo, da se izbrane različice kljub zamenjavam aminokislin v take, ki imajo popolnoma drugačne biokemijske lastnosti, vežejo na holesterol-vsebujoče membrane.

Language:Slovenian
Keywords:Od holesterola odvisni citolizin, perfringolizin O, lipidna membrana, holesterol
Work type:Master's thesis/paper (mb22)
Tipology:2.09 - Master's Thesis
Organization:FKKT - Faculty of Chemistry and Chemical Technology
Year:2021
COBISS.SI-ID:75442179 This link opens in a new window
Views:59
Downloads:10
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Secondary language

Language:English
Title:Characterisation of the amino acid substitutions in cholesterol-binding domain of perfringolysin O
Abstract:
Perfringolysin O (PFO) is an essential virulent factor of the bacterium Clostridium perfringens. It is secreted as a water soluble monomer and binds particularly to cholesterol-rich membranes. Research has showed that the cytolytic mechanism of PFO is initiated by binding of the domain 4 (D4) to the eucaryotic cell membrane and that the cholesterol recognition is mediated by four loops at the tip of the domain. A particular pair of amino acids, T490-L491, termed as cholesterol-recognition motif (CRM) in the literature, is essential for recognizing and binding to cholesterol within the membrane. Despite these findings, the exact mechanism of membrane binding and specific recognition of the membrane cholesterol on a molecular level is not fully understood. The aim of this thesis was to characterize the effects of amino acid substitutions in D4 loops of the protein PFO on binding to various membrane models. We have designed and expressed mutant variations of PFO and individual D4s in the E. coli bacterial expression system and successfully purified them with Ni-NTA affinity chromatography and size-exclusion cromatography. The effect of amino acid substitutions of PFO were characterized with hemolysis assays, whereby the activity of each mutant variation was compared to the activity of the wild type. We discovered that the mutant variations, containing more hydrophobic amino acids in the substituted locations in the CRM, have higher hemolytic activity. Binding assays to model membranes revealed that individual D4s with amino acid substitutions in the proposed CRM bind to multilamelar vesicles, as well as giant unilamelar vesicles with 50 mol. % cholesterol. We have discovered that despite substitutions of amino acids with the kind that have different biochemical properties, certain mutant variations of PFO can bind to cholesterol rich membranes.

Keywords:Cholesterol dependent cytolysin, perfringolysin O, lipid membrane, cholesterol

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