Analysis of protein interactions by microscale thermophoresis : Chromatography demonstrations

Osim svoje kanonske uloge (uspostava kovalentne veze između pripadne tRNASer i aminokiseline serin) seriltRNA- sintetaza (SerRS) ostvaruje i nekanonske uloge djelujući samostalno ili u interakciji s drugim proteinima. U sklopu ovog rada provedena su mjerenja s prethodno identificiranim potencijalnim proteinskim interaktorima SerRS iz biljke Arabidopsis thaliana koristeći mikrotermoforezu, biofizičku metodu za kvantitativnu analizu proteinskih interakcija in vitro. Interakcija SerRS s proteinom do sada nepoznate funkcije (PNF) nije potvrđena, a mikrotermoforeza se nije pokazala prikladnom metodom za identifikaciju interakcije SerRS i glutation-Stransferaze phi 2 (GSTF2). Prethodnim istraživanjima potvrđena je interakcija SerRS i proteina BEN1, koji je uključen u metabolizam brasinosteroidnih hormona. Istraživanja su pokazala da njihovo interakcijsko sučelje čini katalitička domena SerRS-a, te N-terminalni produžetak proteina BEN1. Da bi se dodatno ispitala važnost N-kraja proteina BEN1 metodom mikrotermoforeze ispitana je interakcija SerRS i BEN1 u prisutnosti peptida koji odgovara prvim 17 aminokiselina na N-kraju proteina BEN1. Kompeticijski eksperimenti su pokazali da peptid s N-kraja proteina BEN1 ometa interakciju SerRS:BEN1 čime je potvrđeno da je N-kraj proteina BEN1 doista dio interakcijskog sučelja dva proteina. Kromatografija je zbirni naziv za grupu laboratorijskih tehnika za razdvajanje homogenih smjesa. Uporaba kromatografskih tehnika je učestala te izrazito povezana sa svakodnevnim životom. Zastupljenost kromatografije u osnovnoškolskoj nastavi kemije je izostavljena, a u srednjoškolskoj nastavi se o njoj govori malo. Ovakvo stanje čudi s obzirom na široku primjenu kromatografije kao važne laboratorijske metode. U sklopu ovog rada predložen je 90-minutni nastavni sat koji će poslužiti kao predložak za uključivanje kromatografije u svakodnevnu nastavnu praksu.In addition to its canonical role (the establishment of a covalent bond between the cognate tRNASer and the amino acid serine) seryl-tRNA-synthetase (SerRS) also exhibits noncanonical roles acting alone or in interaction with other proteins. In this diploma thesis, measurements were made with previously identified potential proteins interactors of SerRS from the plant Arabidopsis thaliana using microscale thermophoresis, a biophysical method for the quantitative analysis of protein interactions in vitro. Interaction of SerRS with protein of unknown function (PNF) was not confirmed, while microscale thermophoresis appeared as an inappropriate method for identifying the interaction of SerRS and glutathione S-transferase phi 2 (GSTF2). Previous studies have confirmed the interaction of SerRS and BEN1 protein, which is involved in the brassinosteroid hormones metabolism. Studies have shown that interaction interface consists of SerRS catalytic domain and BEN1 N-terminal extension. To further investigate the importance of N-terminal extension of BEN1 protein, interaction of SerRS and BEN1 was tested in the presence of a peptide corresponding to the first 17 amino acids at the BEN1 N-terminus. Competition experiments have shown that the N-terminal peptide interferes with the SerRS:BEN1 interaction, confirming that BEN1 N-terminal extension is indeed a part of the interaction interface of two proteins. Chromatography is a collective name for a group of laboratory techniques for separating homogeneous mixtures. Elementary school chemistry syllabus lacks knowledge on chromatography while highschool syllabus contains scarce information. This practice is in contrast to wide application of chromatography as an important laboratory method. In this thesis, a 90-minute lesson was proposed that could serve as a template for inclusion of chromatography in regular chemistry teaching practice