Bitran, A.; Bustamante, C.; Marqusee, S.

All proteins can begin to fold on the ribosome, and many proteins critically rely on co-translational folding to attain their native conformation. The molecular details underlying this crucial process, however, remain largely unknown and are not accounted for by structure predictions such as AlphaFold. To probe high-resolution folding during active translation, we develop a novel application of hydrogen-deuterium pulse labeling. We show that two proteins sequentially adopt stable structure during elongation, while a third protein only has time to loosely fold during active elongation. This loose folding kinetically traps the N-terminus and alters the post-translational folding pathway, allowing it to circumvent an aggregation-prone intermediate. These results highlight the crucial non-equilibrium coupling between translation and folding and reveal diverse strategies to promote robust co-translational folding.