Priprema krnjih oblika izoleucil-tRNA-sintetaze 2 iz bakterije Bacillus megaterium i karakterizacija njihove funkcionalnosti

master's thesis

• Title (english): Preparation and functional characterization of truncated forms of isoleucyl-tRNA synthetase 2 from Bacillus megaterium
• Author: Melody Škulac
• Mentor: Ita Gruić Sovulj (mentor)
• Committee member: Ita Gruić Sovulj (predsjednik povjerenstva)
• Committee member: Vesna Petrović Peroković (član povjerenstva)
• Committee member: Iva Juranović Cindrić (član povjerenstva)
• Committee member: Marko Močibob (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Chemistry); Zagreb
• Defense date and country: 2022-12-14, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Chemistry

Abstract

Izoleucil-tRNA-sintetaza (IleRS) je enzim koji katalizira nastajanje izoleucil-tRNAIle u dva
koraka; izoleucin se prvo aktivira uz pomoć ATP-a pa prenosi na tRNAIle
. Enzim posjeduje
modularnu građu s katalitičkom domenom koja je evolucijski najstarija. Izolacija samostojeće
katalitičke domene zato predstavlja prvi korak u pripremi ancestralnih oblika. IleRS spada u
skupinu drevnih i za život presudnih enzima što čini njegovu ancestralnu rekonstrukciju
važnom za razumijevanje evolucije biosinteze proteina, ključnog staničnog procesa. U sklopu
ovog diplomskog rada proizvedena su i kinetički okarakterizirana dva funkcionalno-minimalna
(krnja) oblika IleRS2 iz bakterije Bacillus megaterium nastala uklanjanjem C-terminalne,
korektivne i CP2 domene iz cjelovitog oblika. Topljivost krnjih oblika IleRS bila je niska što
predstavlja veliki izazov za daljnja istraživanja. Kinetička karakterizacija pokazala je da oba
krnja oblika posjeduju aminoacilacijsku aktivnost koja je 3-4 reda veličine manja nego kod
cjelovite IleRS. Dobiveni rezultati ukazuju da su konstruirani krnji oblici IleRS2 dobri modeli
za daljnje istraživanje ancestralnih oblika IleRS.

Abstract (english)

Isoleucyl-tRNA synthetase (IleRS) catalyzes the formation of isoleucyl-tRNAIle in two steps;
ATP-dependent isoleucine activation is followed by isoleucine transfer to the tRNAIle
. The
enzyme is modular with the catalytic domain being the most ancient. Hence, the isolation of a
freestanding catalytic domain represents the first step in the design of IleRS ancestral forms.
IleRS belongs to a group of ancient and life-essential enzymes whose ancestral reconstruction
is relevant for understanding the evolution of protein biosynthesis, a key cellular process.
Within this thesis, we prepared and kinetically characterized two functionally minimal
(truncated) forms of IleRS2 from the bacteria Bacillus megaterium designed by omitting the
C-terminal, editing, and CP2 domains. The solubility of these forms was low, representing a
challenge for further research. Kinetic characterization showed that both forms are active with
3-4 orders of magnitude lower aminoacylation rates compared to full-length IleRS. Our results
suggest these truncated forms are suitable models for further research of IleRS ancestral forms.

• Keywords: aminoaciliranje; Bacillus megaterium; evolucija; izoleucil-tRNA-sintetaza; krnji proteini; pročišćavanje proteina; šaperoni
• Keywords (english): aminoacylation; Bacillus megaterium; evolution; isoleucyl-tRNA synthetase; truncated proteins; protein purification; chaperons
• Language: croatian
• URN:NBN: urn:nbn:hr:217:338820
• Study programme: Title: Graduate university studies in chemistry; specializations in: Research programme; Course: Research programme; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra kemije (magistar/magistra kemije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2022-12-23 09:16:29