Protein domains start to fold co-translationally, while they are being synthesized on the ribosome. Co-translational folding is vectorial and starts in the confined space of the polypeptide exit tunnel of the ribosome. Defects in protein folding cause many human diseases; thus, understanding co-translational folding is of eminent importance. We mapped the folding pathways for two cytosolic proteins of different topology, a small five-helix domain HemK and a five-stranded beta-barrel protein CspA. In solution, both proteins fold by a concerted two-state mechanism. On the ribosome, HemK and CspA start to fold inside the exit tunnel and undergo several structural rearrangements not sampled in solution. HemK folds vectorially by folding and docking of the consecutive α-helical segments. CspA forms compact non-native alpha-helical structures inside and at the vestibule of the exit tunnel and adopts the native fold only after the release from the ribosome. Inner membrane proteins LepB and EmrD insert into the phospholipid bilayer cotranslationally through a dynamic lateral gate of the translocon. We show that the topology of their transmembrane helices is determined at the ribosome due to retention of positive charges at the N-terminus of the transmembrane helix. The results suggest how the ribosome defines the folding landscape of protein domains.

Cotranslational folding of cytosolic and membrane proteins

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