20.500.12556/RUL-184247 Karakterizacija mutanta α-aktinina 1 s povezovalcem med režnjema kalmodulinu podobne domene haracterization of an α-actinin 1 mutant with a linker between the lobes of the calmodulin-like domain Aktin je eden izmed najstarejših in najpogostejših proteinov. Aktinska vlakna imajo v celici številne pomembne vloge, predvsem pri strukturi in premikanju celic. Za regulacijo in povezovanje aktinskih vlaken so odgovorni različni proteini. Eden izmed njih je tudi α-aktinin, prečni povezovalec iz spektrinske družine. Udeležen je pri regulaciji citoskeleta, deljenju in premikanju celice ter mnogih drugih procesih. Sestavljen je iz aktin vezavne domene, paličaste domene in kalmodulinu podobne domene, sestavljene iz EF12 in EF34 poddomen. V človeku poznamo štiri zvrsti α-aktinina, izmed katerih sta dve (ACTN2, ACTN3) na kalcij neobčutljivi in ju najdemo v mišicah, dve (ACTN1, ACTN4) pa na kalcij občutljivi in ju najdemo v vseh celicah. Ob vezavi kalcija na ACTN1 se temu zmanjša sposobnost povezovanja aktina v snope. Trenutno prevladujoča teorija je, da vezava kalcijevega iona na EF12 strukturo proteina stabilizira, hkrati pa povzroči vezavo EF34 na vratno regijo, kar spremeni konformacijo aktin vezavne domene in zmanjša njeno sposobnost povezovanja aktinskih vlaken v snope. Za preverjanje teorije smo izrazili mutanta z insercijo GSGS povezovalca v regijo med EF12 in EF34. Mutantu smo določili afiniteto do vezave kalcijevih ionov, sposobnost povezovanja aktina v snope v prisotnosti kalcija in termostabilnost v prisotnosti in odsotnosti kalcija. Te lastnosti smo nato primerjali s proteinom divjega tipa. Actin is one of the oldest and most common protein on Earth. Actin filaments play many important roles in the cell, especially when it comes to cell structure and movement. Many different proteins are responsible for the regulation and association of the actin network. One of them is α-actinin, a spectrin family crosslinker. It participates in cytoskeleton organization, cell division and movement and many other functions. It consists of an actin-binding domain, rod domain and a calmodulin-like domain, which in turn consists of EF12 and EF34 subdomains. There are four known human α-actinin proteins, of which two (ACTN2, ACTN3) are calcium insensitive and found in muscle, and two (ACTN1, ACTN4) are calcium sensitive and found in all cells. Calcium binding to ACTN1 reduces its ability to bundle actin filaments. The current prevailing theory is that the binding of a calcium ion to the EF12 subdomain stabilizes the protein structure and causes the EF34 subdomain to bind to the neck region, which changes the conformation of the actin-binding domain and reduces its ability to bundle actin filaments. To validate this theory, we have expressed a mutant with a GSGS linker inserted between the EF12 and EF34 subdomains. We determined the mutant′s affinity for calcium ions, actin bundling ability in the presence of calcium and thermostability both in the presence and absence of calcium. We then compared these characteristics to the wild type protein. α-aktinin aktin konformacijske spremembe kalcij α-actinin actin conformational changes calcium true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2026-07-02 12:40:00 2026-07-02 12:40:12 2026-07-03 04:23:36 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 25490 Loborec_Mark_-_Karakterizacija_mutanta_α-aktinina_1_s_povezovalcem_med_reznjema_kalmodulinu_podo.pdf Loborec_Mark_-_Karakterizacija_mutanta_α-aktinina_1_s_povezovalcem_med_reznjema_kalmodulinu_podo.pdf 1 EC25A78AF720765FEFC4A4236F7E4B68 cfca7dbad89eee881c9582d5dbe0cca22faa77208dd0a0481cddeaead89ac17a 4c24dd2a-7602-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=236865 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0