Elucidation of Sequence Constraints for Protein Folding on the Ribosome

Abstract

The pioneering round of protein folding on the ribosome is critical for the proper assembly and function of many proteins. Emerging evidence suggests some proteins sample distinct conformational intermediates during translation, which implies the mechanisms of cotranslational folding may impose specific constraints on protein sequence. To assess how folding on ribosome may shape mutational tolerance, we paired a fluorogenic arrest peptide sensor with deep mutational scanning to survey how 9,480 mutations modulate E. coli dihydrofolate reductase (DHFR) folding on the ribosome in live cells. By controlling for differences in variant abundance, we assess how these mutations specifically impact the efficiency of DHFR folding on stalled ribosomes. Mutagenic trends are broadly correlated with computational estimates for the change in the free energy of the native fold, suggesting that the sequence constraints associated with late-stage folding on the ribosome are broadly similar to those of post-translational folding. However, a close inspection of mutagenic trends reveals that folding on the ribosome is vulnerable to perturbations of specific contacts within a central portion of the adenosine binding domain and the C-terminal {beta}-strands of the discontinuous loop domain. Some of these regions are known to retain structure in the context of conformational intermediates that form on and off the ribosome, while others appear to be specific to cotranslational interactions. Together, our findings highlight how the constraints of folding on the ribosome may constrain evolutionary sequence space.