AP Profiling: Resolving Co-Translational Protein Folding Pathways and Chaperone Interactions In Vivo

Co-translational folding and molecular chaperone action are crucial for productive protein folding in the cell, but how these processes shape folding pathways remains largely unknown. We have utilized translation arrest peptides (APs) to monitor co-translational folding in live bacterial cells, combined with single-molecule optical tweezers experiments, to define the co-translational folding pathway of the GTPase domain (G-domain) from E. coli elongation factor G (EF-G). Surprisingly, the 293 amino acid long domain remains unfolded, without forming stable intermediate structures, until it is fully extruded from the ribosome. The full-length G-domain transitions to its stable native structure via obligate folding intermediates both in isolation and while bound to the ribosome. Folding therefore follows a strictly sequential pathway that initiates at the very C-terminus, which is likely imposed by the structure and topology of the G-domain from EF-G. Consequently, folding and synthesis proceed in opposite directions. G-domains represent a common element in a number of multi-domain proteins. To determine whether their folding pathways are conserved, we have combined our AP approach with cell sorting and deep sequencing into a method that we term "AP profiling". Homologous G-domains exhibited distinct folding patterns that are conserved across distant bacterial species. Individual deletion of the primary nascent chain-binding chaperones, trigger factor and DnaK (Hsp70), resulted in numerous localized changes to co-translational folding while preserving overall G-domain folding, highlighting the functional redundancy of cellular chaperone systems. In summary, we have developed AP profiling as a technique for monitoring co-translational folding in the native cellular environment with high throughput. Combined with single-molecule force spectroscopy, AP profiling yields a unique view of co-translational folding and chaperone function. © FASEB.