Co-translational folding of complex proteins at the single-molecule scale

The folding of proteins into their three-dimensional structures is a critical step for attaining their biological functions. Most folding studies utilize small proteins that refold robustly under experimental conditions. The folding of larger proteins with multiple folding units (domains) has been largely neglected, due to irreversible aggregation and misfolding. Cells reliably produce functional multi-domain proteins in vivo, hinting that critical aspects contributing to their folding are overlooked during traditional ensemble experiments. Co-translational folding, ribosomal interactions, and chaperone binding coordinate to result in reliable folding of multi-domain proteins. Since multi-domain proteins comprise a majority fraction of all proteomes, knowledge of the factors contributing to their folding will enrich our understanding of protein biogenesis. This thesis aims to characterize processes contributing to efficient biogenesis of multi-domain proteins and to develop techniques to achieve this goal. I used single-molecule manipulation with optical tweezers to study co-translational folding. This approach resolves folding at high resolution and avoids ensemble averaging and aggregation. I first discuss the development of a novel protocol for generating proteins for optical tweezers experiments. This workflow greatly reduces the preparative time for optical tweezers experiments and is especially useful for disordered proteins, which are difficult to prepare biochemically. Using this and other approaches, I have dissected the co-translational folding pathway of the multi-domain protein elongation factor G (EF-G). I found that co-translational folding benefits EF-G by preventing otherwise prevalent inter-domain misfolding. Chaperone interactions extend the beneficial folding window for EF-G during translation and prevent a novel detrimental interaction between folded and unfolded domains. The folding of E. coli phosphoglycerate kinase (ecPGK), another multi-domain protein, does not exhibit inter-domain misfolding that hinders folding. Rather, refolding often stalls at an intermediate formed by the N-terminal domain. My findings indicate how co-translational folding helps to overcome this kinetic bottleneck. The ribosome stabilizes the N-terminal domain, which contrasts with the mostly destabilizing consequences of ribosome-nascent protein interactions reported so far. This stabilization likely occurs through interactions with the helical linker that connects ecPGK domains. Taken together, the data presented in this thesis have revealed critical features of co-translational folding that contribute to efficient protein biogenesis