Analysis of protein interactors of cytosolic seryl-tRNA synthetase from plant Arabidopsis thaliana

Seril-tRNA-sintetaze (SerRS) su enzimi odgovorni za uspostavu kovalentne veze između pripadne tRNA i aminokiseline serin čime ostvaruju podlogu za ispravnu biosintezu proteina. Uz ovu kanonsku ulogu, SerRS proteini imaju i nekanonske uloge koje ostvaruju samostalno ili uspostavljajući interakcije s drugim staničnim proteinima. U sklopu ovog rada, za potrebe biofizičkih mjerenja pripremljeni su prethodno identificirani potencijalni interakcijski partneri citosolne SerRS iz biljke Arabidopsis thaliana i to glutation-S-transferaza phi 2 (GSTF2) i protein do sada nepoznate funkcije (PNF), dok je kodirajući slijed za ribosomski protein L9 uspješno ugrađen u plazmidne vektore. Nadalje, u svrhu određivanja interakcijskog sučelja citosolne SerRS i nedavno otkrivenog interakcijskog partnera, proteina BEN1 uključenog u metabolizam brasinosteroidnih hormona, osmišljene su i pripremljene skraćene varijante navedenih proteina. Mikrotermoforetska mjerenja pokazala su da citosolna SerRS uspostavlja interakciju s proteinom BEN1 svojom središnjom, katalitičkom domenom, dok protein BEN1 za interakciju s proteinom SerRS koristi nestrukturirani hidrofilni produžetak na N-kraju. Također je pokazana i važnost GST-privjeska koji značajno poboljšava prinos i stabilnost katalitičke domene citosolne SerRS.Seryl-tRNA synthetases (SerRS) are enzymes responsible for covalent attachment of cognate tRNA with amino acid serine thus creating a foundation for accurate protein biosynthesis. In addition to this canonical role, SerRSs participate in other noncanonical pathways either independently or via interactions with other cellular proteins. In this thesis, recently identified potential interaction partners of the cytosolic SerRS from plant Arabidopsis thaliana, glutathione-S-transferase phi 2 (GSTF2) and protein of unknown function (PNF) were prepared for biophysical analysis, while gene encoding ribosomal protein L9 was successfully cloned in plasmid vectors. Furthermore, in order to determine interaction surface between cytosolic protein SerRS and recently identified interaction partner, protein BEN1 involved in brassinosteroid hormone metabolism, truncated variants of these proteins were designed. Microthermophoresis measurements have shown that cytosolic SerRS interacts with the metabolic protein BEN1 using its central catalytic domain, while protein BEN1 interacts with SerRS using its N-terminal unstructured hydrophilic extension. In addition, significant improvement in yield and stability of the catalytic domain of cytosolic SerRS using the GST-tag was also observed