The bacterial GTPase Obg is critical for ribosome maturation, translational control and stress adaptation, making it an attractive target for the de novo design of protein binders amid the growing threat of antimicrobial resistance. Using the advanced generative model RFdiffusion, we designed about 200 candidates and evaluated them with bioinformatic methods. This is how we reduced the selection to four binders with the highest likelihood of forming a stable complex with Obg. We successfully expressed and purified recombinant Obg and prepared the four selected binders, of which only one proved suitable for further analyses – two candidates were excluded due to unsuccessful IVA cloning, the third due to insufficient protein concentration for continued work. In SEC analysis of the complex, we observed changes in Obg’s elution profile upon addition of the binder, indicating an interaction. From the obtained chromatogram we inferred that the interaction induced a structural change in Obg, shifting the complex peak towards higher elution volumes. Although additional structural confirmation testing was not performed due to time constraints, the results supported the hypothesis that RFdiffusion enables successful design of functional binders that form stable complexes with the target. In a broader context, the thesis highlighted the important microbiological and immunological challenge of therapeutically resistant microbes and emphasized the need for new approaches to develop agents against resistant pathogens.
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