Primerjava metod za optimizacijo predkristalizacijskih pogojev proteinov na primeru lizocima

Kobale, Anja (author), Turk, Dušan (mentor), Dolinar, Marko (comentor)

Strukture proteinov lahko določamo na različne načine. Mednje spada tudi rentgenska difrakcija. Le-ta zahteva makromolekulske kristale, v katerih se molekule geometrijsko uredijo, kar omogoča sipanje rentgenskih žarkov, ki je pogoj za uspešno določitev 3D strukture. Na urejenost makromolekul v kristalu vpliva tudi mikrookolje, v katerem se makromolekule nahajajo in kjer bo potekala kristalizacija. Mikrookolje je sestavljeno iz raztopine proteina in raztopine rezervoarja, ki predstavlja spojine z različnimi načini delovanja. Raziskave so pokazale, da tudi raztopina, v kateri je raztopljen protein, preden pripravimo kristalizacijske kapljice, pomembno prispeva h končnemu rezultatu – proteinskim kristalom. V tej magistrski nalogi smo se posvetili predkristalizacijskim pogojem testnega proteina lizocima. Primerjali smo dve metodi, s katerima lahko dobimo optimalne predkristalizacijske pogoje, diferenčno dinamično fluorimetrijo (DSF) in topnostnim testom. Namen je bil iz združenih rezultatov poiskati ugodnejše pogoje za kristalizacijo, kot jih dobimo z vsako metodo posebej, saj vsaka od obeh metod deluje na svoj način in ima svoje zahteve. Opravili smo eksperimente z obema metodama, nato rezultate obeh primerjali, jih združili in nastavili kristalizacijo, da bi preverili kristalizacijo proteina pri izbranih pogojih. Pripravili smo tudi odločitvena drevesa, kdaj slediti kateri izmed metod glede na tip proteina, ki ga preiskujemo, razpoložljivo količino proteina ter opremo. Iz rezultatov smo ugotovili, da kadar združimo najboljše rezultate obeh metod, ni zagotovo, da bo lizocim kristaliziral, saj so se pojavili kristali lizocima tako v pufrskih pogojih, ki so bili teoretično najboljši, kot v pufrskih pogojih, ki so bili teoretično najslabši. Iz rezultatov te naloge sklepamo, da nobena izmed metod ne more razkriti analiziranih pogojev, za katere lahko z gotovostjo trdimo, da bo pri njih lizocim kristaliziral.

• Language: Slovenian
• Keywords: predkristalizacijski pogoji, diferenčna dinamična fluorimetrija (DSF), topnostni test, kristalizacija
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2021
• PID: 20.500.12556/RUL-129742
• COBISS.SI-ID: 79048451
• Publication date in RUL: 07.09.2021

Secondary language

• Language: English
• Title: Comparison of methods for optimization of the precrystallization buffer in the case of lysozyme

Abstract

Protein structures can be determined by using different approaches. One of them is X-ray diffraction which requires the proteins to form macromolecular crystals diffracting X-rays which in turn could lead to a successful determination of the 3D structure. The arrangement of the macromolecules in a crystal is affected by the microenvironment. The microenvironment consists of protein and reservoir solutions. According to the literature the solution in which the protein is present before being introduced in the crystallization droplets, importantly contributes to the end result – growth of protein crystals. This master's thesis focuses on the precrystallization conditions of the test protein lysozyme. We compared two different methods which give optimal precrystallization conditions, fluorescence monitoring of thermal denaturation of proteins (DSF) and solubility test. The aim was to find favorable conditions for crystallization than obtained by each method separately because both methods work by their own principles and have their own requirements. The experiments were carried out using both methods. After comparing the results, we set up the crystallization to investigate the protein crystallization at selected conditions. To choose the appropriate method according to the protein investigated, its available quantity and accessible equipment, a decision tree was designed. We discovered that even when we combine the best results of the two methods, it is not certain that lysozyme will crystallize. Namely, lysozyme crystals appeared both in buffer conditions that were theoretically best and in buffer conditions that were theoretically worst. The crystallization results indicate that none of the methods can reveal the best conditions for which we could say for certain that lysozyme will crystallize.

• Keywords: precrystallization conditions, differential scanning fluorimetry (DSF), solubility test, crystallization