Projektbeschreibung
Entschlüsselung der Co-Co-Verbindung
Bei der Verbindung von Komponenten, die in verschiedenen Phasen zusammengefügt werden, entsteht eine Co-Co-Verbindung. Co-Co-Verbindungen kommen hauptsächlich bei der Bildung homomerer Oligomere zum Einsatz und sie kommen in allen Lebensbereichen vor. Bisher ist jedoch sehr wenig darüber bekannt, welche molekularen Mechanismen sie antreiben. Das EU-finanzierte Projekt Co-coAssembly wird die Mechanismen der Co-Co-Verbindung mithilfe der dimeren chorismatischen Mutase als repräsentativen Kandidaten aus einem Hochdurchsatz-Screening für Co-Co-verbindende Proteinkomplexe in E. coli untersuchen. Unter Einsatz einer speziellen Tomografie steht bei der Studie die dreidimensionale Anordnung der E. coli-Ribosomen im Mittelpunkt, um herauszufinden, wie die Organisation des Translationsapparats die Co-Co-Verbindung ermöglicht.
Ziel
Almost all fundamental biological processes involve protein complexes and therefore, efficient folding and assembly of homo- and hetero-oligomers is critical for cellular functionality and integrity. Recent studies have shown that many protein complexes assemble co-translationally by one fully-synthesized subunit engaging another subunit that is still in nascent state (co-post assembly). An ongoing study in the Bukau lab now revealed that assembly can also occur by interaction of two partner nascent chains (co-co assembly). Co-co assembly is mostly employed for the formation of homo-oligomers and exists in all kingdoms of life..
Despite initial evidence of its existence, very little is known about the molecular mechanisms driving co-co assembly. This includes information on whether co-co assembly requires co-localization of two polysomes or can happen on one polysome.. Furthermore, it is currently unclear whether co-co interactions require preceding nascent chain folding steps and to what extent co-translationally acting chaperones coordinate the process and the impact of translation speed on co-co assembly.
I propose to study mechanisms of co-co assembly using the dimeric chorismate mutase (PheA) as a representative top candidate from a high throughput screen for co-co assembling protein complexes in E. coli. Employing cryo-electron tomography, I will analyse the three dimensional arrangement of E. coli ribosomes in the context of a polysome to assess how organization of translational machinery allows co-co assembly. Moreover, I will study the co-translational cascade of folding steps of chorismate mutase by utilizing FRET on in vitro prepared nascent chains. Finally, I plan to explore the impact of co-translationally acting chaperones and translation kinetics on co-co assembly, by performing disome-selective profiling analysis in chaperone mutant cells lacking Trigger Factor and DnaK and in mutants that synthesize proteins with reduced translation kinetics.
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MSCA-IF - Marie Skłodowska-Curie Individual Fellowships (IF)Koordinator
69117 Heidelberg
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