Abstract
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-β Ig 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.
Highlights
Proteins that fold cotranslationally may do so in a restricted configurational space, due to the volume occupied by the ribosome
To what extent is the cotranslational folding pathway of a protein influenced by the presence of the ribosome and by the vectorial emergence of the polypeptide chain during translation? Recent studies have shown that small proteins can fold inside the ribosome exit tunnel [1], while other proteins can fold at the mouth of the tunnel [2]; some proteins may be too large to fold within the confines of the ribosome (e.g., DHFR) [3]
Results from all three techniques show that I27 folds in the mouth of the ribosome exit tunnel; our simulations correctly capture the onset of folding in I27 and three mutant variants, allowing us to predict how destabilization of regions that fold early and late in the isolated domain affect folding on the ribosome
Summary
We have explored the hypothesis that the broad force peak of mutant M67A might be due to interactions between an exposed hydrophobic cavity on I27[M67A] resulting from the mutation and hydrophobic surface residues of ribosomal proteins uL23 and uL29 By introducing such interactions into the model, we are able to obtain a broad peak in the force profile very similar to that seen in experiment (Fig. 4D). The simulations suggest that formation of native contacts between the N and C termini is somewhat more important when folding takes place in the mouth of the exit tunnel (L = 31 residues) than far outside the ribosome (L = 51 residues) (Fig. 5 D–F, upper left-hand corners) This is likely due to the greater difficulty of forming these contacts (examples are shown in Fig. 5 G–I) under ribosomal confinement; forming them becomes more critical in enabling the protein to fold
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