diplomsko delo
Ajda Cafun (Author), Kristina Djinović Carugo (Mentor)

Abstract

α-aktinin-1 je aktin-vezavni protein, ki na območju stresnih vlaknih in fokalnih stikov prečno povezuje aktinske filamente ter jih pripenja na membranske adhezijske komplekse. S tem opravlja pomembno vlogo pri procesu celičnega premikanja. Njegova funkcija povezovanja aktinskih filamentov je negativno regulirana s povišano znotrajcelično koncentracijo kalcija. Vezava kalcijevega iona v kalmodulinu podobno domeno človeškega α-aktinina-1 povzroči konformacijske spremembe, ki vplivajo na dinamiko priležne aktin-vezavne domene (ABD). Natančnejši mehanizem regulacije in z njim povezane konformacijske spremembe še niso pojasnjeni, zato smo želeli okarakterizirati spremembe v dinamiki domene ABD med apo (z vezanim Ca2+-ionom) in holo (brez vezanega Ca2+-iona) oblikama α-aktinina-1 z metodo NMR. Ker je celoten α-aktinin-1 prevelik za preučevanje z NMR, želimo za nadaljnje raziskave pripraviti segmentno označen protein, pri katerem bo izotopsko označena zgolj domena ABD. Pripravo segmentno označenega proteina omogoča ligacija dveh ločeno izraženih fragmentov željenega proteina z metodo trans-spajanja preko razcepljenega inteina. V tem delu smo postavili izhodišče za pripravo segmentno označenega α-aktinina-1. Uspešno smo izolirali inteinska konstrukta z zapisom za fragmenta α-aktinina-1 in ju ligirali z metodo trans-spajanja preko razcepljenega inteina Cfa. Ligacijo inteinskih konstruktov smo optimizirali in pokazali, da je izkoristek ligacije največji pri temperaturi 33 °C. Z ligacijo inteinskih konstruktov v velikemmerilu smo potrdili, da je izkoristek ligacije dovolj velik za pripravo segmentno označenega α-aktinina-1 za meritve NMR.

Keywords

citoskelet;proteini;alfa-aktinin-1;aktin-vezavna domena;NMR spektroskopija;segmentno izotopsko označevanje;trans-spajanje proteinov;PTS;diplomska dela;

Data

Language: Slovenian
Year of publishing:
Typology: 2.11 - Undergraduate Thesis
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [A. Cafun]
UDC: 577.112(043.2)
COBISS: 1538424515 Link will open in a new window
Views: 577
Downloads: 168
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Other data

Secondary language: English
Secondary title: Optimization of ligation of intein constructs for the preparation of segmentally labeled α-actinin-1
Secondary abstract: α-actinin-1 is a cytoskeletal actin-binding protein found in stress fibers and focal adhesion sites, where it crosslinks actin filaments and attaches them to membrane junction complexes. Consequently, it plays an important role in cell migration. Its crosslinking function is thought to be negatively regulated by increased intracellular calcium levels. Upon binding of acalcium ion, calmodulin-like domain of human α-actinin-1 undergoes structural changes that also affect the dynamics of juxtaposed ABD domain. The exact mechanism of regulation and consequent conformational changes remain elusive. Therefore, we wish to characterise changes in the dynamics of ABD domain between calcium-free and calcium-bound formsof α-actinin-1 using NMR spectroscopy. Since α-actinin-1 is too large to be successfully analysed by NMR, we wish to prepare segmentally labeled protein where only ABD domain is isotopically labeled. Two separately expressed fragments could be ligated via protein trans-splicing using split inteins to form segmentally labeled protein. In this work we thus set the basis for the preparation of segmentally labeled α-actinin-1. We successfully isolated two intein constructs, each containing a fragment of split α-actinin-1 and ligated them via trans-splicing, using split intein Cfa. We also optimized the ligation of intein constructs and showed that the highest yield is obtained at temperature 33 °C. With a large-scale ligation of intein constructs we confirmed that the yield is high enough for the preparation of segmentally labeled α-actinin-1for NMR studies.
Secondary keywords: alpha-actinin-1;actin binding domain;NMR spectroscopy;segmental isotopic labeling;PTS;
Type (COBISS): Bachelor thesis/paper
Study programme: 1000371
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, UNI Biokemija
Pages: 51 str.
ID: 11220347