magistrsko delo
David Titovšek (Author), Kristina Djinović Carugo (Mentor), Brigita Lenarčič (Thesis defence commission member), Miha Pavšič (Thesis defence commission member)

Abstract

α-aktinin-1 je eden izmed najpomembnejših proteinov, ki prečno povezujejo aktinske filamente v stresnih vlaknih in fokalnih stikih. Vsebuje 4 motive EF-roke, ki skupaj tovorijo kalmodulinu podobno domeno (CaMD), a le EF1 lahko veže kalcijev ion. Raziskave kažejo, da vezava iona Ca2+ na α-aktinin-1, zmanjša njegovo sposobnost prečnega povezovanja aktinskih filamentov in predstavlja način regulacije njegovega delovanja. Nedavno je bil na podlagi dosedanjih raziskav oblikovan model regulacije α-aktinina-1 s kalcijem. Ta predpostavlja, da ob vezavi iona Ca2+ α-aktinin-1 zasede bolj zaprto konformacijo, kjer se CaMD veže na povezovalni peptid med vezavno domeno za aktin in domeno s spektrinskimi ponovitvami. To α-aktininu-1 onemogoči povezovanje aktinskih filamentov v paralelne ali antiparalelne snope, a natančen mehanizem na molekularni ravni še ni poznan. Da bi ovrednotili predlagani model, smo izvedli omejeno proteolizo divjega tipa α-aktinina-1 (zaprta oblika) in mutanta z onemogočeno vezavo kalcijevih ionov (odprta oblika). Proteoliza s tripsinom je pokazala, da je α-aktinin-1 bolj odporen na razgradnjo v prisotnosti kalcijevih ionov, kar se sklada s predpostavljenim modelom. Tudi pri proteolizi s termolizinom smo opazili razlike v cepitvenem vzorcu med holo (z vezanim Ca2+) in apo (brez vezanega Ca2+) obliko, a vezave CaMD na povezovalni peptid na podlagi rezultatov nismo mogli ovrednotiti, zato so za potrditev modela potrebni nadaljnji eksperimenti. Da bi dodatno ovrednotili konformacijske spremembe na molekularnem nivoju, smo pripravili konstrukte polovičnega dimera α-aktinina-1 in njegove mutante z zmanjšano površinsko entropijo za kristalizacijo. Proteinske kristale holo (s Ca2+) in apo (brez Ca2+) oblike α-aktinina-1 smo pripravili z mutantom polovičnega dimera z zmanjšano površinsko entropijo (mutaciji K79A in E81A). Pridobljeni kristali bodo omogočili določitev kristalne strukture α-aktinina-1 z in brez vezanega Ca2+ z rentgensko difrakcijo.

Keywords

aktinski citoskelet;aktin vezavni proteini;naddružina spektrina;alfa-aktinin-1;kalcij;konformacijske spremembe;omejena proteoliza;kristalizacija;magistrska dela;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [D. Titovšek]
UDC: 577.112(043.2)
COBISS: 85704451 Link will open in a new window
Views: 233
Downloads: 45
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Other data

Secondary language: English
Secondary title: Conformational changes of human α-actinin-1 upon binding of calcium ions
Secondary abstract: α-actinin-1 is a major actin filament cross-linking protein in stress fibers and focal adhesions. It contains 4 EF-hand motifs (together they form the calmodulin-like domain – CaMD – of α-actinin-1), but only EF1 is able to bind calcium. Research suggests that Ca2+ binding to α-actinin-1 weakens its ability to cross-link actin filaments and thus acts as a regulator of its function. Recently, the model of regulation of actin-binding activity of α-actinin-1 was proposed, whereupon calcium binding α-actinin-1 obtains a more closed conformation, in which CaMD interacts with the neck region between actin-binding domain and spectrin region. This prevents α-actinin-1 from cross-linking actin filaments into parallel or anti-parallel bundles, but the exact molecular details have not yet been determined. To evaluate the proposed model, we performed limited proteolysis assays of full-length wild type α-actinin-1 (closed form) and a full-length α-actinin-1 mutant with impaired Ca2+ binding ability (open form). Proteolysis with trypsin showed that α-actinin-1 is more resistant to digestion in the presence of Ca2+ (closed form), which is in agreement with the proposed model. Proteolysis with thermolysin also revealed some differences in the cleavage pattern between Ca2+-bound and -free α-actinin-1, but we were unable to characterize the binding of CaMD to the neck region, therefore further experiments are needed to validate the proposed model. To further characterize the conformational changes at the molecular level, we prepared half-dimer of α-actinin-1 and its mutants with reduced surface entropy for crystallization. We have produced protein crystals of the holo (with Ca2+) and apo (without Ca2+) forms of the reduced surface entropy half-dimer construct (mutations K79A and E81A), which will be used to determine the crystal structure of Ca2+-bound and -free α-actinin-1 by X-ray diffraction.
Secondary keywords: alpha-actinin-1;calcium;conformational changes;limited proteolysis;crystallization;
Type (COBISS): Master's thesis/paper
Study programme: 1000377
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, smer Biokemija
Pages: 65 str.
ID: 13534228