Microscale Thermophoresis Platform Comprehensively Analyzes Bacterial Translation Initiation, Quantifying Antibiotic and Antimicrobial Peptide Effects

In prokaryotes, translation initiation is a complex, dynamic process essential for protein synthesis, involving the ribosome, mRNA, initiator tRNAfMet, and initiation factors (IFs). Traditional analytical methods, often relying on hazardous radioactive labeling or surface immobilization, present significant safety risks and methodological constraints, limiting comprehensive, real-time molecular interrogation. There is a critical need for a unified, safe, and highly sensitive platform to dissect these intricate interactions and facilitate the discovery of novel antimicrobial agents targeting this fundamental pathway.

Researchers developed a fluorescence-based analytical platform integrating microscale thermophoresis (MST) to comprehensively interrogate bacterial translation initiation. They systematically applied MST to a panel of fluorescently labeled components, including initiator tRNAfMet, various mRNAs, and initiation factors. To expand the fluorescence toolbox for ribosomal studies, a robust BODIPY-labeling protocol was developed for 70S ribosomes, with structural integrity and function confirmed via nano differential scanning fluorimetry, stopped-flow kinetic assays, and peptide-synthesis activity tests. The platform was then used to investigate the effects of compounds like streptomycin and rumicidin-1 on translation initiation.

The developed MST platform successfully quantified assembly pathways and equilibria as bacterial translation initiation progressed from simple bimolecular interactions to higher-order, multicomponent complexes. It facilitated probing the crucial roles of magnesium ions and initiation factors upon 30S initiation complex formation. The platform's utility was demonstrated by investigating the effects of various compounds, including antibiotics and antimicrobial peptides. Specifically, the antibiotic streptomycin's dissociation constant (Kd) was determined for both 30S and 70S ribosomes, proving identical at 0.3 ± 0.1 μM. This precise quantification highlights the platform's capability for detailed mechanistic studies. Furthermore, the platform effectively demonstrated the impact of the antimicrobial peptide rumicidin-1 on translation initiation, showcasing its versatility for novel compound discovery. > The antibiotic streptomycin dissociation constant for both 30S and 70S ribosomes was determined to be identical at 0.3 ± 0.1 μM.