Summary
Proteins that fold cotranslationally do so in a restricted configurational space, due to the volume occupied by the ribosome. Here, we investigate the cotranslational folding of an all-β immunoglobulin domain, titin I27, whose intrinsic folding mechanism has been extensively characterized. 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 ribosome, by essentially the same pathway as free I27, with only subtle shifts of critical contacts from the C to the N terminus.
