20.500.12556/RUL-169009 Opredelitev deleža sekundarne strukture in termostabilnosti transkripcijskega faktorja PHOX2B in njegovih domen Defining the proportion of secondary structures and thermostability of the transcription factor PHOX2B and its domains Odkritje, da se nekatere celične strukture vedejo kot kapljice in delujejo kot brezmembranski organeli, je eno izmed prelomnih odkritij v sodobni biologiji. Brezmembranski organeli nastanejo preko fazne separacije tekoče-tekoče in so običajno obogateni s proteini, ki vsebujejo dolge neurejene regije z nizko kompleksnostjo, kar pomeni, da se v sekvenci pojavlja le nekaj tipov aminokislinskih ostankov. V nalogi smo raziskovali vlogo polialaninske regije v človeškem proteinu PHOX2B in skušali ugotoviti, kako ta nizkokompleksni motiv vpliva na strukturo in obnašanje proteina. S pomočjo orodja Alphafold 3.0 smo preko različnih kombinacij ugotavljali strukturo in interakcije med nativnim proteinom ter med proteinom in zaporedjem DNA, na katero se veže. Ugotovili smo, da protein večinoma nima urejene strukture, z izjemo α-vijačnice DNA-vezavne domene Homeobox. Nekatere patološke mutacije uvedejo energijsko neugodne interkacije na DNA domeni ter tako onemogočijo vezavo proteina na promotorsko regijo, vendar ne povzročijo strukturnih sprememb proteina. Pri eksperimentalnem delu smo izražali tri verzije proteina PHOX2B. To so bili DNA-vezavna domena omenjenega proteina, C-končna domena in celoten protein. Posneli smo jim CD-spekter pred in po termični denaturaciji ter jim določili termično stabilnost. Tako smo potrdili, da sta celoten protein in izolirana C-končna domena pretežno neurejena oziroma brez sekundarne strukture. Pri termični denaturaciji se izkaže, da se nobena izražena različica ne vrne popolnoma v nativno stanje, ampak ostane v še bolj neurejeni obliki. The discovery that some cellular structures behave like droplets and mimic the structure of membraneless organelles is one of the major breakthroughs in modern biology. These structures are formed with the liquid-liquid phase separation and are usually enriched with proteins that contain uncomplexed unstructured regions, which means that the sequence contains low diversity of amino acids. We have studied the role of the polyalanine region in the human protein PHOX2B and tried to discover how this uncomplexed motif has an impact on the structure and the behavior of the protein. With the help of Alphafold 3.0 and trying different combinations I looked at the structures and interaction between the native protein itself and with its binding DNA. I figured out that the protein mostly is unstructured, with the exception of the α-helical structure of the Homeobox DNA binding domain. A number of pathologic mutations change some energetically favourable interactions to unfavourable, which inhibit the binding of the transcription factor to the promotor sequence but it does not change the structure of the protein itself. Experimentally we produced the expression of three versions of the protein PHOX2B. Those were the isolated DNA-binding domain, the isolated C-terminal region of the protein and the whole protein with no mutations. We recorded their CD specters before and after thermal denaturation and we also analysed their thermal stability. With that, we confirmed that the native protein PHOX2B and its C-terminal domain are mostly unstructured and without secondary structures. After thermal denaturation we can see that neither the whole protein nor any shorter version of the protein has the same secondary structure as before, but they are now even more unstructured. PHOX2B polialaninska regija CD spektrometrija Alphafold fazna separacija tekoče-tekoče PHOX2B polyAla region CD spectrometry Alphafold liquid-liquid phase separation true false false Slovenski jezik Angleški jezik Diplomsko delo/naloga 2025-05-07 08:55:01 2025-05-07 08:55:09 2025-05-09 13:10:20 0000-00-00 00:00:00 2025 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 24131 235386371 Novel_Matija_-_Karakterizacija_faze_separacije_tekoce-tekoce_v_proteinu_z_alaninsko_regijo.pdf Novel_Matija_-_Karakterizacija_faze_separacije_tekoce-tekoce_v_proteinu_z_alaninsko_regijo.pdf 1 0689B4B6EF0F5AC6F5A13A0D3BDF17E6 a77dee513fe2482276bcac89b83eabfb5e77e016beddf879b2dc5425d3e44dee 0608a2d0-2b0f-11f0-b232-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=210114 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0