Energetic dependencies dictate folding mechanism in a complex protein.

Large proteins with multiple domains are thought to fold cotranslationally to minimize interdomain misfolding. Once folded, domains interact with each other through the formation of extensive interfaces that are important for protein stability and function. However, multidomain protein folding and the energetics of domain interactions remain poorly understood. In elongation factor G (EF-G), a highly conserved protein composed of 5 domains, the 2 N-terminal domains form a stably structured unit cotranslationally. Using single-molecule optical tweezers, we have defined the steps leading to fully folded EF-G. We find that the central domain III of EF-G is highly dynamic and does not fold upon emerging from the ribosome. Surprisingly, a large interface with the N-terminal domains does not contribute to the stability of domain III. Instead, it requires interactions with its folded C-terminal neighbors to be stably structured. Because of the directionality of protein synthesis, this energetic dependency of domain III on its C-terminal neighbors disrupts cotranslational folding and imposes a posttranslational mechanism on the folding of the C-terminal part of EF-G. As a consequence, unfolded domains accumulate during synthesis, leading to the extensive population of misfolded species that interfere with productive folding. Domain III flexibility enables large-scale conformational transitions that are part of the EF-G functional cycle during ribosome translocation. Our results suggest that energetic tuning of domain stabilities, which is likely crucial for EF-G function, complicates the folding of this large multidomain protein.