INVESTIGATION OF THE MECHANISM OF RESCUER 4, A DE NOVO PROTEIN SEQUENCE

Abstract

One of the goals of synthetic biology is to isolate novel protein sequences that support essential biological functions. By using both rational and combinatorial design, the Hecht group has successfully discovered multiple synthetic sequences capable of sustaining life. Most of these functional de novo sequences form stable, well-folded protein structures, but the Rescuer 4 protein enables the survival of auxotrophic ∆metC E. coli cells in minimal media despite being disordered and insoluble. Preliminary experimental results suggested that the Rescuer 4 protein may rescue ∆metC through gene regulation, by co precipitating with MetJ, a transcriptional repressor protein. In order to investigate the rescue mechanism of Rescuer 4, multiple variants of the sequence were created through site-directed mutagenesis. These sequences were then transformed into ∆metC cells for life-or-death screening and growth analysis. Most variants exhibited reduced or abolished rescue function, verifying that the protein—rather than the RNA—is responsible for the rescue of ∆metC and confirming the interaction between MetJ and Rescuer 4 plays a key part in the mechanism. In addition, a variant sequence with enhanced rescue efficiency was discovered. Proteomics analysis of this strain points to an upregulation of chaperone proteins. These findings provide further insight into the mechanism of Rescuer 4 and demonstrate how de novo proteins can interact with natural biomolecules to support life-sustaining functions.