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Kinetika vezave ekvinatoksina II na lipidne kapljice in velike unilamelarne vezikle
ID Vončina, Blaž (Avtor), ID Maček, Peter (Mentor) Več o mentorju... Povezava se odpre v novem oknu

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Izvleček
Ekvinatoksin II (Eqt II) je proteinski toksin iz konjske morske vetrnice (Actinia equina), ki ga uvrščamo v družino aktinoporinov. Vetrnicam služi za plenjenje in obrambo pred plenilci. Eqt II tvori kationsko selektivne pore v tarčnih lipidnih membranah. Proces tvorjenja pore vključuje vezavo Eqt II na sfingomielin (SM), ki se nahaja v lipidnih membranah, vstavitev N-terminalne α-vijačnice v membrano, oligomerizacijo ter nastanek pore. Namen naloge je bil preučiti razliko med vezavo Eqt II na lipidni dvosloj, ki ga vsebujejo veliki unilamelarni vezikli (LUV), in vezavo na lipidni monosloj, ki ga vsebujejo lipidne kapljice (LK). Preučevali smo tudi vpliv N-terminalne α-vijačnice na začetno vezavo toksina. Že pripravljene plazmide z zapisom za mutanta Eqt IIV8C,K69C smo namnožili v bakterijskem sevu Escherichia coli DH5α. Ekspresijo s plazmidom vstavljenih genov smo inducirali z dodatkom 0,5 M izopropil-β-D-tiogalaktopiranozida v bakterijsko kulturo. S pomočjo stresanja, soniciranja in centrifugiranja smo iz bakterijske usedline izolirali Eqt IIV8C,K69C. Tega smo nato očistili z ionsko-izmenjevalno in gelsko kromatografijo ter ga skoncentrirali z ultrafiltracijo. Pripravili smo reducirano različico mutanta Eqt IIV8C,K69C, pri katerem se lahko N-terminalna α-vijačnica prosto giblje, ter oksidirano različico mutanta, pri kateri je gibanje N-terminalne α-vijačnice onemogočeno zaradi disulfidnega mostička, ki nastane med jedrom molekule in N-terminalno α-vijačnico. Koncentracije lipidov v pripravljenih LUV in LK smo določili s pomočjo kompletov za določanje koncentracije lipidov. Eksperiment smo izvedli na fluorimetru z napravo za hitro pomešanje reagentov. Ob vgradnji triptofanskih ostankov (ti se nahajajo na vezavnem delu molekule Eqt II) v nepolarno membrano pride do povečanja fluorescence. Izmerjene spremembe intenzitete fluorescence smo opisali z matematičnim modelom psevdo-prvega reda reakcij adsorpcije. K eksperimentalnim krivuljam smo prilegali monoeksponentne krivulje. Ob vezavi reduciranega mutanta Eqt IIV8C,K69C, tako na LUV kot tudi na LK, smo izmerili večjo intenziteto maksimalne fluorescence kot ob vezavi oksidiranega mutanta Eqt IIV8C,K69C. Menimo, da je to posledica negibljive N-terminalne α-vijačnice pri oksidiranemu mutantu, ki predstavlja oviro, zaradi katere se triptofanski ostanki v lipidno membrano vgradijo manj globoko. Ob vezavi na LUV (tako v primeru oksidiranega kot tudi reduciranega mutanta Eqt IIV8C,K69C) smo izmerili nekoliko večje intenzitete maksimalne fluorescence kot ob vezavi na LK. To kaže na nekoliko večjo dostopnost SM v dvoslojni lipidni membrani LUV kot SM v lipidnem monosloju LK. Večja sposobnost vezave Eqt II na LUV bi lahko bila tudi posledica nekoliko večjega razmerja v koncentracijah med holesterolom in SM v LUV v primerjavi z istim razmerjem v LK, saj membranski holesterol ojača vezavo aktinoporinov na SM. Bistvenih razlik med začetno vezavo Eqt II na LUV in LK nismo odkrili.

Jezik:Slovenski jezik
Ključne besede:ekvinatoksin II, veliki unilamelarni vezikli, lipidne kapljice, sfingomielin, lipidni dvosloj, lipidni monosloj, holesterol, hemoliza, triptofanska fluorescenca, pora, actinia equina
Vrsta gradiva:Magistrsko delo/naloga
Organizacija:BF - Biotehniška fakulteta
Leto izida:2017
PID:20.500.12556/RUL-99311 Povezava se odpre v novem oknu
COBISS.SI-ID:4566607 Povezava se odpre v novem oknu
Datum objave v RUL:12.01.2018
Število ogledov:1342
Število prenosov:494
Metapodatki:XML RDF-CHPDL DC-XML DC-RDF
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Sekundarni jezik

Jezik:Angleški jezik
Naslov:Kinetics of the equinatoxin II binding on lipid droplets and large unilamelar vesicles
Izvleček:
Equinatoxin II is a toxin from a protein family of actinoporins . It was found in beadlet anemone (Actinia equina), which uses it to prey and to defend itself from predators. Eqt II forms cation selective pores in target lipid membranes. The pore-forming process includes binding of Eqt II to sphingomyelin (SM) in lipid membranes, insertion of N-terminal α- helix into the membrane, oligomerization and eventual formation of pore. The purpose of our research was to examine the differences between toxin's binding on lipid bilayer of large unilamelar vesicles (LUV) and lipid monolayers of lipid droplets (LK). We also examined the role of N-terminal α-helix in the initial binding to lipid membrane. We multiplied plasmids, containing information for Eqt IIV8C,K69C mutant, in Escherichia coli DH5α strain. The Eqt IIV8C,K69C was then isolated by ion-exchange and gel chromatography. The oxidised and reduced forms of the toxin mutant were prepared. N-terminal α-helix of the oxidized form of Eqt IIV8C,K69C is unable to move because of the cysteine bond between the helix and the core of the molecule. N-terminal α-helix of reduced form of Eqt IIV8C,K69C can move freely. The concentrations of lipids in prepared LUV and LK were determined by the use of special kits for determining concentration of lipids. In order to study the binding of Eqt II on lipid membranes, we used stopped flow fluorescence spectroscopy. When tryptophan residues (that are present in the binding site of the Eqt II) are inserted into the nonpolar lipid membrane, the intensity of fluorescence increases. To describe the changes in fluorescence intensity we used the pseudo-first order rate equation. We fitted monoexponential curves to the experimental curves. When reduced form of Eqt IIV8C,K69C was bound to either LUV or LK, we measured higher values of maximal fluorescence intensity than in the case of oxidized form of Eqt IIV8C,K69C. The latter has the N-terminal α-helix connected to the core of the molecule, which disables its movement. We suppose that this rigid structure poses a hindrance that disables deeper insertion of tryptophan residues into the lipid membrane, which results in a bit lower maximal fluorescence intensity. Higher values of maximal fluorescence intensities were also measured when either oxidized of reduced form of Eqt IIV8C,K69C was bound on LUV as when it was bound to LK. We suppose that SM in the membranes of lipid bilayer of LUV might be a bit more accessible than SM in the lipid monolayer of LK. However, the greater binding ability of Eqt II to LUV might be the result of a higher cholesterol to SM ratio in LUV than it is in LK. Cholesterol in cell membranes is known to increase the binding ability of actinoporins to SM. We did not discover significant differences between binding of Eqt II to LUV and LK.

Ključne besede:equinatoxin II, large unilamelar vesicles, lipid droplets, sphingomyelin, lipid bilayer, lipid monolayer, cholesterol, hemolysis, tryptophan fluorescence, pore, actinia equina

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