The Folding Pathway of an Ig Domain is Conserved On and Off the Ribosome

Proteins that fold cotranslationally may do so in a restricted configurational space, due to the volume occupied by the ribosome. How does this environment, coupled with the close proximity of the ribosome, affect the folding pathway of a protein? Previous studies have shown that the cotranslational folding process for many proteins, including small, single domains, is directly affected by the ribosome. Here, we investigate the cotranslational folding of an all-b immunoglobulin domain, titin I27. Using an arrest peptide-based assay and structural studies by cryo-EM, we show that I27 folds in the mouth of the ribosome exit tunnel. Simulations that use a kinetic model for the force-dependence of escape from arrest, accurately predict the fraction of folded protein as a function of length. We used these simulations to probe the folding pathway on and off the ribosome. Our simulations - which also reproduce experiments on mutant forms of I27 - show that I27 folds, while still sequestered in the mouth of the ribosome exit tunnel, by essentially the same pathway as free I27, with only subtle shifts of critical contacts from the C to the N terminus. Significance Statement Most proteins need to fold into a specific three-dimensional structure in order to function. The mechanism by which isolated proteins fold has been thoroughly studied by experiment and theory. However, in the cell proteins do not fold in isolation, but are synthesized as linear chains by the ribosome during translation. It is therefore natural to ask at which point during synthesis proteins fold, and whether this differs from the folding of isolated protein molecules. By studying folding of a well characterized protein domain, titin I27, stalled at different points during translation, we show that it already folds in the mouth of the ribosome exit tunnel, and that the mechanism is almost identical to that of the isolated protein.