magistrsko delo
Povzetek
Egerolizini so relativno majhni proteini (15–20 kDa) z značilnim zvitjem s
prevladujočimi β-strukturami. Predstavniki družine so prisotni v mnogih taksonomskih
skupinah, največ pa jih najdemo pri glivah. Kljub relativno široki taksonomski
porazdelitvi je le nekaj egerolizinov natančno raziskanih.
Biološka vloga egerolizinov pri glivah še vedno ni popolnoma razjasnjena. Trenutno
obstaja le hipotetičen nabor možnih bioloških vlog. Glede na znane podatke verjetno
delujejo med rastjo in razvojem organizma ali igrajo pomembno vlogo pri obrambi in
preživetju organizmov. Značilnost nekaterih egerolizinov je, da delujejo hemolitično in
citolitično, a v nekaterih primerih do aktivnosti pride šele ob vezavi partnerskega proteina
MACPF. Opažena je bila še sposobnost vezave lipidov, pri tem pa se specifičnost vezave
na določeno vrsto lipida med različnimi egerolizini razlikuje.
V sklopu tega dela smo obravnavali štiri egerolizine z neobičajnim N-končnim
podaljškom iz različnih glivnih organizmov. Zanimivi so predvsem zato, ker so na
podlagi zaporedja drugačni kot večina ostalih egerolizinov. Raziskovali smo, kako Nkončni podaljšek vpliva na strukturo in funkcijo egerolizinov. Rezultate smo primerjali z
znanim egerolizinom OlyA (ostreolizin A) in z aktinoporinom EqtII (ekvinatoksin II),
katerega N-končno zaporedje z motivom α-vijačnice pomembno vpliva na njegovo
citolitično aktivnost.
Z bioinformacijsko analizo smo napovedali in preučili strukturne modele tarčnih
egerolizinov ter nekatere druge biokemijske lastnosti. Predvideli smo, da imajo štirje
egerolizini: fusver, fusgra, phiatt in traver podaljšan N-konec z napovedano strukturo αvijačnice. Ta je pri egerolizinih fusver, fusgra, phiatt amfipatična in bi lahko služila
vezavi v membrano. Pripravili smo vse štiri egerolizine in pokazali, da noben od njih ni
hemolitičen, niti se ne veže na različne liposome. Za namen določitve strukture, smo
egerolizine poskusili kristalizirati, kar nam v izbranem naboru kristalizacijskih pogojev
ni uspelo.
Ključne besede
podaljšan egerolizin;ostreolizin A;ekvinatoksin II;lipidne membrane;bioinformacijska analiza;magistrska dela;
Podatki
Jezik: |
Slovenski jezik |
Leto izida: |
2021 |
Tipologija: |
2.09 - Magistrsko delo |
Organizacija: |
UL FKKT - Fakulteta za kemijo in kemijsko tehnologijo |
Založnik: |
[A. Uzar] |
UDK: |
577.112(043.2) |
COBISS: |
97991683
|
Št. ogledov: |
224 |
Št. prenosov: |
23 |
Ocena: |
0 (0 glasov) |
Metapodatki: |
|
Ostali podatki
Sekundarni jezik: |
Angleški jezik |
Sekundarni naslov: |
Preparation and biochemical characterization of recombinant aegerolysins with an N-terminal extension |
Sekundarni povzetek: |
Aegerolysins are relatively small proteins (15–20 kDa) with characteristic β-structural
fold. Representatives of the aegerolysin protein family are found in many taxonomic
groups; however, they are most commonly found in fungi. Despite a relatively broad
taxonomic distribution, only a few aegerolysin-like proteins have been thoroughly
researched.
The biological role of aegerolysins in fungi is still not fully elucidated. Currently, there
is only a hypothetical set of possible biological roles. According to known data, they are
likely to act during the growth and development of the organism and/or they play an
important role in the defence and survival of the organisms. Some aegerolysins are known
to have hemolytic and cytolytic activity, but in most cases that activity occurs only upon
interaction with a partner MACPF-domain protein. The ability to bind lipids has also been
observed, but the specificity of binding to a particular type of lipid varies between
representative proteins.
As part of this work, we considered four fungal aegerolysins with a specific N-terminal
extension. They are interesting mainly because of their difference from most aegerolysins
in terms of the sequence. We investigated how the N-terminal extension affects the
structure and function of aegerolysins. The results were compared to the known
aegerolysin OlyA (ostreolysin A) and to actinoporin EqtII (equinatoxin II), whose Nterminal α-helix structure significantly affects its cytolytic activity.
Using bioinformaticc analysis, we predicted and studied structural models of target
aegerolysins and some other biochemical properties. We predicted that four aegerolysins:
fusver, fusgra, phiatt, and traver have an elongated N-terminus with a predicted α-helix
structure. This is amphipathic in the case of aegerolysin fusver, fusgra, phiatt and could
potentially bind to the membrane. We prepared and purified all four aegerolysins and
showed that none of them were hemolytic, nor did they bind to different liposomes. For
the purposes of determining the structure, we tried to crystallize aegerolysins. We were
not able to achieve the aegerolysins crystallization in the selected set of crystallization
conditions. |
Sekundarne ključne besede: |
extended aegerolysin;lipid membranes;bioinformatic analysis;ostreolysin A;equinatoxin II;Beljakovine;Univerzitetna in visokošolska dela; |
Vrsta dela (COBISS): |
Magistrsko delo/naloga |
Študijski program: |
1000377 |
Konec prepovedi (OpenAIRE): |
1970-01-01 |
Komentar na gradivo: |
Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, smer Biokemija |
Strani: |
86 str. |
ID: |
14503999 |