Načrtovanje, izražanje in ovrednotenje heterodimernih obvitih vijačnic na osnovi tropomiozina

Mežnaršič, Eva (author), Bratkovič, Tomaž (mentor), Križnik Gradišar, Helena (co-mentor)

Dobro poznavanje pravil zvijanja različnih naravnih proteinov nam omogoča načrtovanje proteinskih nanostruktur, kakršnih ne najdemo v naravi, de novo. Nove strukture lahko uporabimo v medicini, kemiji in biotehnologiji, npr. za ciljano dostavo zdravil v tkiva, razvoj cepiv, tkivno inženirstvo, načrtovanje biosenzorjev, biokatalizo in razvoj bionanomaterialov. Za načrtovanje de novo dovolj dobro poznamo le nekaj manjših naravnih domen proteinov, ki jih lahko uporabimo kot module za sestavljanje večjih poliedrskih nanostruktur. Proteinski origami je primer modularne topološko pogojene nanostrukture, sestavljene iz obvitih vijačnic, ki predstavljajo module. Posamezni moduli interagirajo izključno s svojim načrtovanim partnerjem in se tako zvijejo v načrtovano strukturo. Obvite vijačnice so polipeptidna zvitja dveh verig, za katera je značilen vzorec sedmih aminokislinskih ostankov, imenovanih heptade. V sklopu magistrskega dela smo načrtovali, izrazili in ovrednotili tri obvite vijačnice: Tropo-Cn6/Tropo-Dn6, Tropo-Cn12/Tropo-Dn12 in Tropo-Cn14/Tropo-Dn14. Te so del pete generacije heterodimernih obvitih vijačnic, dolgih 15 heptad, na osnovi tropomiozina, načrtovanih na Kemijskem inštitutu. V peti generaciji smo izbrali najbolj obetaven par iz četrte generacije in uvedli spremembe v devet prej nemodificiranih heptad. Velikost načrtovanih polipeptidov smo določili z metodo dinamičnega sipanja svetlobe (DLS) in ugotovili, da se je skladala s predvideno velikostjo okoli 15 nm. Delež sekundarne vijačne strukture smo ugotavljali z metodo cirkularnega dikroizma (CD). Pri paru Tropo-Cn6/Tropo-Dn6 v nasprotju s pričakovanji nismo opazili vijačnih struktur. Para Tropo-Cn12/Tropo-Dn12 in Tropo-Cn14/Tropo-Dn14 sta dala boljše rezultate. Z merjenjem temperaturne stabilnosti sekundarnih vijačnih struktur smo ugotovili, da je pri paru Tropo-Cn12/Tropo-Dn12 večja razlika med Tm posameznega polipeptida in Tm para. Glede na rezultate smo se zato odločili, da bomo nadaljnjo optimizacijo heterodimernih obvitih vijačnic na osnovi tropomiozina nadaljevali na podlagi Tropo-Cn12/Tropo-Dn12.

• Language: Slovenian
• Keywords: obvita vijačnica, tropomiozin, proteinski origami, načrtovanje de novo
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2019
• PID: 20.500.12556/RUL-111805
• Publication date in RUL: 14.10.2019

Secondary language

• Language: English
• Title: Design, production and characterization of tropomyosin based coiled-coil dimers

Abstract

Good understanding of rules governing folding of natural proteins is the basis for de novo design of protein nanostructures. These new structures can be applied in medicine, chemistry and biotechnology, i. e. for targeted drug delivery, vaccine design, tissue engineering, sensor design, biocatalysis and development of bionanomaterials. Only a few smaller domains are known well enough for de novo design. These can serve as modules for the design of polyhedric nanostructures. Protein origami is an example of modular topological nanostructure comprised from coiled coils that represent modules. The polypeptide folds into the desired structure due to each module interacting exclusively with its partner. Coiled coils are polypeptide folds comprised of two chains that are characterized by a pattern of seven amino acid residues called heptads. We designed, expressed and characterized three pairs of coiled coil forming polypeptides: Tropo-Cn6, Tropo-Dn6, Tropo-Cn12, Tropo-Dn12, Tropo-Cn14 and Tropo-Dn14. These were designed at National Institute of Chemistry in Ljubljana as a part of fifth generation of 15 heptads long tropomyosin-based coiled coil heterodimers. In the fifth generation we chose the most promising pair from the fourth generation of coiled coils and introduced changes into nine previously unmodified heptads. The size of designed polypeptides was determined by dynamic light scattering (DLS). The measurements matched the predicted size of 15 nm. We also determined the presence of secondary structures by circular dichroism (CD). Against our expectations, the pair Tropo-Cn6/Tropo-Dn6 did not show any secondary structures. Pairs Tropo-Cn12/Tropo-Dn12 and Tropo-Cn14/Tropo-Dn14 produced better results. Measurements of the thermal stability of the secondary structures displayed bigger difference in Tm between individual polypeptide and the designed pair with pair Tropo-Cn12/Tropo-Dn12 than Tropo-Cn14/Tropo-Dn14. We decided that further optimization of tropomyosin-based coiled coil heterodimers will thus continue on the basis of the Tropo-Cn12/Tropo-Dn12.

• Keywords: coiled coil, tropomyosin, protein origami, de novo design