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Vpeljava tehnike predstavitve na ribosomih za proučevanje interakcij proteinov z membranami
ID Omersa, Neža (Author), ID Anderluh, Gregor (Mentor) More about this mentor... This link opens in a new window

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Abstract
Od holesterola odvisni citolizini (ang. cholesterol-dependent cytolysins, CDC) imajo pomembno vlogo pri virulentnosti bakterij. Razumevanje njihovega delovanja je pomembno za razvoj učinkovin za zaviranje njihove aktivnosti ter za možnost njihove uporabe v terapevtske, biotehnološke in sinteznobiološke namene. S tehniko predstavitve na ribosomih smo pridobili vezavno proteinsko ogrodje na osnovi racionalno načrtovanega proteina z ankirinskimi ponovitvami (DARPin-a, ang. designed ankyrin repeat protein), ki se je vezalo na Y406A, ki je od pH odvisen mutantni CDC. Izbrani DARPin se je z veliko afiniteto (KD ~ 100 nM) vezal na Y406A na tak način, da je še vedno omogočal vezavo Y406A na membrano, vendar do nastanka pore ni prišlo, hkrati pa je bila vezava DARPin-a na Y406A mogoča, ko je bil slednji že vezan na membrano. Z vpeljavo tehnike predstavitve na ribosomih smo pridobili tudi različice domene CDC za vezavo na membrano (PFOD4), ki so se na membrano vezale z različnimi vezavnimi parametri. Divji tip PFOD4 se je vezal na membrano s 50 molarnimi odstotki holesterola z afiniteto (KD) v nanomolarnem območju. Testirali smo dve različici PFOD4, od katerih se je ena vezala na membranski holesterol manj specifično, in sicer na dve različno dostopni populaciji membranskega holesterola. Obe različici sta imeli KD v enakem območju kot divji tip PFOD4. Na osnovi selektivnega in specifičnega zaviranja aktivnosti CDC z DARPin-om smo postavili proteinska logična vrata na membrani in pokazali princip delovanja takega sistema v sintezni biologiji. Tehnika predstavitve na ribosomih se je izkazala kot primerna tudi za pridobitev različic membranskovezavnih domen CDC s spremenjenimi vezavnimi lastnostmi. V kombinaciji z uveljavljenimi tehnikami za merjenje interakcij predstavlja predstavitev na ribosomih dobrodošlo dodatno orodje za proučevanje interakcij proteinov z membranami, pri čemer so njene glavne prednosti predvsem prilagodljivost, omogočanje številnih kombinacij zamenjav aminokislinskih ostankov ter možnost zorenja afinitete.

Language:Slovenian
Keywords:predstavitev na ribosomih, DARPin, CDC, holesterolna specifičnost, površinska plazmonska resonanca, usmerjena evolucija
Work type:Doctoral dissertation (mb31)
Organization:MF - Faculty of Medicine
Year:2020
Publication date in RUL:22.10.2020
Views:330
Downloads:76
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Secondary language

Language:English
Title:Implementation of ribosome display technique for studying protein-membrane interactions
Abstract:
Cholesterol-dependent cytolysins (CDCs) are important virulence factors. An in-depth understanding of their membrane binding and pore formation mechanism is crucial for the development of their inhibitors and for their utilization in therapeutic, biotechnological, and synthetic biology applications. The ribosome display technique enabled us to gain the protein binding scaffold based on designed ankyrin protein (DARPin) with a high binding affinity (KD ~ 100 nM) towards the Y406A, which is a pH-dependent mutant of CDC. Selected DARPin bound to the Y406A and inhibited its pore forming activity, although the membrane-binding step of Y406A was not affected. The DARPin was able to bind to the Y406A also when the Y406A was already inserted into a membrane. Implementation of ribosome display was successful also for selection of CDC membrane-binding domain variants (PFOD4) with different binding parameters. The wild-type PFOD4 bound to the membrane with 50 molar percent of cholesterol with a nanomolar affinity (KD). Between the two tested PFOD4 variants, one bound to the membrane cholesterol less specifically, namely to two differently accessible populations of membrane cholesterol, whereas both variants had the KDs in a similar range as the wild-type PFOD4. Based on selective and specific inhibition of the CDC with the DARPin, we constructed the protein logic gate on a membrane and showed a proof of concept for such a system in synthetic biology. Ribosome display proved appropriate also for enrichment of membrane-binding domains with altered binding characteristics. Together with established techniques for measuring molecular interactions, ribosome display is an appreciated additional tool for studying protein-membrane interactions, with its main advantages being flexibility, allowance of numerous combinations of amino acid substitutions, and the possibility of affinity maturation.

Keywords:ribosome display, DARPin, CDC, cholesterol specificity, surface plasmon resonance, directed evolution

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