doktorska disertacija
Žiga Strmšek (Author), Helena Gradišar (Mentor), Roman Jerala (Co-mentor)

Abstract

Glavna prednost polipeptidih nanostruktur, v primerjavi z večino ostalih polimerov, je tvorba zelo kompleksnih struktur, ki jih določa zaporedje aminokislin. Narava je tekom evolucije raziskala le majhen del teoretično možnih proteinskih zvitij, medtem ko večji del t.i. proteinskega vesolja ostaja neraziskan. Pristopov k razvoju in raziskovanju proteinskih zvitij, ki v naravni niso prisotna, je več. Eden od pristopov k racionalnemu načrtovanju de novo proteinskih zvitij je proteinski origami na osnovi ovitih vijačnic. Proteinski origami iz ovitih vijačnic je načrtovan po principu modularnosti in daljnosežnih interakcij med segmenti ovitih vijačnic. Za to proteinsko zvitje so značilne raznolike poliedrske kletke, pri katerih stranice tvorijo ovite vijačnice, ki so med seboj povezane z gibljivimi proteinskimi povezovalci in s hidrofilno votlino na sredini nanokletke. V doktorskem delu smo raziskali strategije predstavitve proteinskih domen na ogliščih tetraedrične nanokletke z direktno genetsko fuzijo. Delovanje razvitega sistema smo preizkusili s predstavitvijo ene (GFP), dveh (GFP in RFP) ali štirih (štiri GFP ali RFP) proteinskih domen. Na podlagi in silico modeliranja in analize sipanja rentgenskih žarkov pri nizkih kotih se je pokazalo, da je proteinska domena, predstavljena na četrtem oglišču kot fuzija na C-koncu verige, gibljiva. Z zamenjavo mesta fuzije znotraj zaporedja TET12SN smo jo prestavili v notranjost polipeptidne verige in tako zmanjšali njeno gibljivost. Po učvrstitvi vseh štirih domen smo pripravili serijo sedmih konstruktov z različnimi razmerji GFP in RFP, pri katerih smo dokazali uspešno predstavitev proteinskih domen z analitsko gelsko filtracijo in s sipanjem rentgenskih žarkov pri nizkih kotih. TET12SN-RRRR, tetraedrično proteinsko ogrodje, dekoriranim s štirimi RFP proteinskimi domenami, smo uspešno uporabili kot cepivo za predstavitev antigenov. Miši, imunizirane s TET12SN-RRRR so razvila IgG protitelesa proti antigenu RFP hitreje in bolj zanesljivo kot miši, ki so bile imunizirane z monomernim antigenom. Kot alternativno strategijo predstavljanja proteinskih domen na proteinskem origamiju smo TET12SN dekorirali s SpyCatcher/SpyTag proteinskimi domenami, katere smo uporabili za izgradnjo centralnega tetraedra, dekoriranega z (i) enim, (ii) dvema ali (iii) tremi ligandi vključenimi v tetraedrične proteinske kletke. Trenutno sta velikost in kompleksnost poliedričnih proteinskih origamijev omejena z številom dostopnih ortiginalnih ovitih vijačnic. Da bi se izognili tej omejitvi, smo raziskali če lahko uspešno načrtujemo tetraedričen proteinski origami, pri katerem isti par ovitih vijačnic uporabimo večkrat. Dokazali smo, da isti par ovitih vijačnic znotraj tetraedrične nanokletke lahko uporabimo največ dvakrat, ter na tak način uspešno načrtovali in okarakterizirali tetraedrično nanokletko s ponovljenimi pari ovitih vijačnic enkrat (TET12SN(2CC)), dvakrat (TET12SN(22CC)) in trikrat (TET12SN(222CC)). Da bi dokazali modularnost razvitega sistema predstavljanja proteinskih domen na ogliščih, smo pripravili serijo sedmih konstruktov z različnimi razmerji GFP in RFP, le da smo za osnovno ogrodje uporabili TET12SN(22CC). Uspešno izolirane proteine iz te serije smo okarakterizirali z analitsko gelsko filtracijo in sipanjem rentgenskih žarkov pri nizkih kotih, medtem ko smo konstrukt dekoriran z štirimi rdečimi fluorescentnimi proteini še dodatno vizualizirali z krio-elektronsko mikroskopijo. Predstavljen sistem predstavljanja proteinskih domen na ogliščih proteinskih origamijev ima potencial za uporabo v medicinske, industrijske in biotehnološke namene.

Keywords

molekularna genetika;proteinski origami;proteinske domene;doktorske disertacije;Sintezna biologija;Disertacije;Molekularna biologija;Proteinske domene;Zvijanje beljakovin;

Data

Language: Slovenian
Year of publishing:
Typology: 2.08 - Doctoral Dissertation
Organization: UL MF - Faculty of Medicine
Publisher: [Ž. Strmšek]
UDC: 577.2(043.3)
COBISS: 131519747 Link will open in a new window
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Other data

Secondary language: English
Secondary title: Presentation of protein domains on the scaffold of coiled-coil protein origami tetrahedron
Secondary abstract: Compared to other biopolymers, polypeptide nanostructures are able to form highly complex structures that are defined by the aminoacid sequence. Nature explored only a small portion of all protein folds, while huge regions of 'protein space' remain unexplored. There are several approaches to discovering or creating new protein folds – coiled-coil protein origami (CCPO) is one of such. CCPO is de novo rationally designed protein fold from linked coiled-coil (CC) forming modules. CCPO is modular, composed of CCs that form the edges around the internal hydrophilic cavity and can form diverse polyhaedral shapes. In the presented thesis we explored the strategies of presenting protein domains at the vertices of tetrahedral CCPOs by direct genetic fusion. As a proof of principle, four tetrahedral CCPO (TET12SN) constructs were decorated either with one green fluorescent protein (GFP), one green and one red fluorescent protein (RFP) or four GFPs or four RFPs. All constructs expressed well and were characterised by small-angle X-ray scattering (SAXS), which, coupled with in silico modelling, revealed that there was some undesired flexibility of the fourth presented protein domain. The mobility was reduced by modifying the insertion site – and to further explore the developed system, seven additional constructs, with different ratios of GFP : RFP were prepared and analysed with SAXS and analytical gel filtration. Moreover, TET12SN-RRRR, tetrahedral CCPO decorated with four RFPs, was used as a vaccine for antigen presentation. Mice immunised with an antigen, presented on a scaffold (TET12SN-RRRR), developed IgG antibodies against RFP faster and with higher titer compared to mice immunised with a monomeric antigen. As an alternative strategy of protein domain grafting onto the CCPO scaffold, TET12SN was decorated with SpyCatcher/SpyTag protein domains, that were used to create bigger protein complexes of a tetrahedron decorated with (i) one, (ii) two or (iii) three additional ligand tetrahedral CCPO. Currently, the complexity of polyhaedral CCPOs are limited by the number of available orthogonal CCs. We investigated if the same CC pair could be used several times within the same structure. We successfully designed and characterised a tetrahedral CCPO with one (TET12SN(2CC)), two (TET12SN(22CC)) or three (TET12SN(222CC)) CC pair that occured two times in the sequence. In order to prove that protein domains could still be presented on CCPO with repeated CC pairs, seven protein constructs with varying ratios of GFP : RFP were created utilizing newly designed CCPO TET12SN(22CC). All successfully isolated proteins from the last series were also characterized by an analytical gel filtration and SAXS; moreover, protein construct with four RFPs was characterised in more detail with cryoelectron microscopy. The developed system for presenting protein domains at the vertices of tetrahedral CCPO offers a powerful tool in expanding toolkit of protein origami, that could one day be used in medical, industrial and/or biotechnological applications.
Secondary keywords: Molekularna biologija;Beljakovine;Stereokemija;Univerzitetna in visokošolska dela;
Type (COBISS): Dissertation
Study programme: 0
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Medicinska fak.
Pages: 113 str., 12 str. pril.
ID: 13292547