Drevni proteini kao termički stabilniji analozi postojećih proteina

master's thesis

• Title (english): Ancestral proteins as thermostable analogues of extant proteins
• Author: Marija Viher
• Mentor: Marko Močibob (mentor)
• Committee member: Marko Močibob (predsjednik povjerenstva)
• Committee member: Marijana Đaković (član povjerenstva)
• Committee member: Ivan Kodrin (član povjerenstva)
• Committee member: Ita Gruić Sovulj (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Chemistry); Zagreb
• Defense date and country: 2017-09-25, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Chemistry

Abstract

Aminoacil-tRNA-sintetaze kataliziraju spajanje aminokiseline s pripadnom tRNA. Leucil-tRNA-sintetaza sadrži dodatnu domenu za popravak pogreške za hidrolizu nepripadno sparenih supstrata. Računalno rekonstruirani drevni proteini često su termički stabilniji od proteina iz kojih su izvedeni. Međutim, do sada ih se istraživalo zbog proučavanja mehanizama evolucije, a ne zbog primjene njihove termičke stabilnosti. Predmet ovoga rada su drevna leucil-tRNA-sintetaza i njezina mutirana inačica s inaktiviranom domenom za popravak. Termička stabilnost drevne leucil-tRNA-sintetaze ispitana je dvjema različitim metodama: diferencijalnom pretražnom fluorimetrijom i mjerenjem aktivnosti enzima pri povišenoj temperaturi. Određeni su njezini kinetički parametri u uvjetima ustaljenog i predustaljenog stanja. Drevna leucil-tRNA-sintetaza je termostabilnija od leucil-tRNA-sintetaze iz E. coli, a enzimi su identični u reakcijama aktivacije, prijenosa aminokiseline i popravka pogreške. Drevni enzimi stoga mogu pronaći primjenu kao termički stabilniji analozi postojećih proteina u uvjetima gdje je poželjna veća stabilnost enzima.

Abstract (english)

Aminoacyl-tRNA synthetases catalyse coupling of amino acids with their cognate tRNAs. Leucyl-tRNA synthetase has additional editing domain in which hydrolysis of misactivated amino acids takes place. Inferred ancestral proteins often show greater thermostability than their extant descendant proteins. However, until now they were used in evolution mechanism research and not due to application of their thermal properties. Subject of this thesis are ancestral leucyl-tRNA synthetase and its mutant with inactivated editing domain. Thermostability of ancestral leucyl-tRNA-synthetase was monitored with two different methods: differential scanning fluorimetry and enzyme activity measurement. Its kinetical parameters were determined with steady state and pre-steady state kinetics. Ancestral leucyl-tRNA synthetase is more thermostable than E. coli leucyl-tRNA synthetase and both enzymes are kinetically identical in reactions of amino acid activation, transfer on tRNA and editing. Therefore, ancestral enzymes can be used as thermostable analogues of extant proteins in conditions that ask for greater stability.

• Keywords: diferencijalna pretražna fluorimetrija; drevna leucil-tRNA-sintetaza; kinetika predustaljenog stanja; kinetika ustaljenog stanja; kružna mutageneza; termička stabilnost
• Keywords (english): ancient leucyl-tRNA-synthetase; circular mutagenesis; differential scanning fluorimetry; pre-steady state kinetics; steady state kinetics; thermostability
• Language: croatian
• URN:NBN: urn:nbn:hr:217:702721
• 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: 2017-10-17 16:39:07