20.500.12556/RUL-170809 Merjenje kinetike disociacije dolgih na novo načrtovanih obvitih vijačnic Measuring the dissociation kinetics of long de novo designed coiled-coils Raziskovali smo dolge, de novo načrtovane obvite vijačnice osnovane na naravnem proteinu tropomiozinu. Zaradi svoje modularnosti in specifičnega povezovanja imajo velik potencial pri razvoju nanomaterialov ter v biomedicinskih aplikacijah. Širši namen naloge je prenesti princip izpodrivanja verige z oprimkom iz sveta DNA na obvite vijačnice. Osredotočili smo se na tropomiozinski par C12, D12 in njune različice, ki smo jih uspešno izrazili, izolirali in očistili. S kombinacijo metod kromatografije z ločevanjem po velikosti z večkotnim detektorjem sipanja svetlobe (SEC-MALS), cirkularnim dikroizmom (CD) in izotermično titracijsko kalorimetrijo (ITC) smo potrdili njihovo molekulsko maso, sekundarno strukturo in afiniteto vezave. Z metodo razdeljene luciferaze NanoLuc smo določili konstanto disociacije. Z metodama prenosa energije z resonanco fluorescence (FRET) in razdeljene luciferaze NanoLuc smo pokazali potencialno izpodrivanje obvitih vijačnic s pomočjo oprimka, kar potrjuje, da imajo obvite vijačnice potencial za načrtovanje dinamičnih proteinskih sistemov. We investigated long, de novo designed coiled-coils based on natural tropomyosin. Due to their modularity and specific binding, they offer great potential in the development of nanomaterials and biomedical applications. From the world of DNA nanotechnology, we wanted to transfer the principle of toehold mediated strand displacement to coiled-coils. We focused on the tropomyosin pair C12, D12 and their variants, which we successfully expressed, isolated and purified. Using a combination of size-exclusion chromatography with multi-angle light scattering (SEC-MALS), circular dichroism (CD) and isothermal titration calorimetry (ITC), we confirmed their molecular weight, secondary structure and binding affinity. Using the NanoLuc split luciferase method, we determined the dissociation constant. Using fluorescence resonance energy transfer (FRET) and split luciferase NanoLuc methods, we also demonstrated the possibility of partial toehold mediated strand displacement of coiled-coils, confirming that coiled-coils have the potential for the design of dynamic protein systems. obvite vijačnice proteinska kinetika de novo načrtovanje proteinov coiled-coils protein kinetics de novo protein design true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2025-07-17 07:15:21 2025-07-17 07:15:28 2025-07-19 03:40:29 0000-00-00 00:00:00 2025 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 257827 242876675 49472.pdf 49472.pdf 1 A50483C4A5B885A03EB54B56C883C20D ad47f969675877d19270bef524cc6da249ff6a076143a366d48cd69a616ced98 1bbff3ea-62cb-11f0-b232-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=213018 Biotehniška fakulteta 0 0 0