A small single-domain protein folds through the same pathway on and off the ribosome

Abstract

In vivo, proteins fold and function in a complex environment subject to many stresses that can modulate a protein's energy landscape. One aspect of the environment pertinent to protein folding is the ribosome, since proteins have the opportunity to fold while still bound to the ribosome during translation. We use a combination of force and chemical denaturant (chemomechanical unfolding), as well as point mutations, to characterize the folding mechanism of the src SH3 domain both as a stalled ribosome nascent chain and free in solution. Our results indicate that src SH3 folds through the same pathway on and off the ribosome. Molecular simulations also indicate that the ribosome does not affect the folding pathway for this small protein. Taken together, we conclude that the ribosome does not alter the folding mechanism of this small protein. These results, if general, suggest the ribosome may exert a bigger influence on the folding of multidomain proteins or protein domains that can partially fold before the entire domain sequence is outside the ribosome exit tunnel.protein folding | ribosome | cotranslational folding | single-molecule force spectroscopy | optical tweezers

The folding and unfolding behavior of ribonuclease H on the ribosome

“…The correlation between the three-state and two-state folding of variants in standard refolding experiments and their ability to read through the SecM stall implies that the intermediate observed in CD and HDX studies off the ribosome is likely populated on the ribosome and again suggests that the general folding trajectory, or pathway, of RNH* is unaltered on the ribosome. These data, together with the pulse proteolysis results, agree with studies of the folding mechanisms of other small proteins on the ribosome, which have shown that the folding trajectory does not change for RNCs relative to free proteins (5,11). Combining this pulse proteolysis approach with other site-specific mutations in a f-value analysis will further elucidate the folding pathway of ribosome-tethered RNH* I53D and could be applied to other nascent chains to characterize the effect of the ribosome on a range of folding mechanisms.…”

“…This accounts for the observed decrease in stability for these RNCs. Although we can only sample a limited range of urea concentrations in these RNC experiments, the similarity in urea dependence (m ‡ unf) indicates the protein is traversing the same transition state barrier on and off the ribosome, consistent with observations for the small proteins titin and SH3 (5,11). Assuming that RNH* I53D folds in a two-state mechanism on the ribosome (populating only U and N), this implies that the presence of the ribosome does not affect the folding rate and suggests a mechanism by which the ribosome destabilizes the nascent chain by promoting its unfolding.…”

“…From our pulse-proteolysis experiments, we infer an off-ribosome folding rate of 0.1 s -1 for RNH* I53D, relative to kfold = 0.74 s -1 for RNH* off the ribosome in bulk CD experiments (27). These questions might be best answered by simulations, which can dissect the mechanism of force generation (5,11,25).…”

“…Both of these proteins have complex folding pathways, including the formation of transient intermediates, and both show differences between their cotranslational and refolding pathways. However, folding pathways are not necessarily altered by the ribosome; two small beta-sheet domains, the src SH3 domain and titin I27, show simple two-state folding and appear to fold via the same pathway both on and off the ribosome (5,11). Is populating transient folding intermediates required for folding to be modulated by the ribosome?…”

Activation of a Protein Kinase Via Asymmetric Allosteric Coupling of Structurally Conserved Signaling Modules

“…We attached DNA handles via thiol chemistry to two cysteines engineered at specific positions in the protein (see Methods) 13,14 . The handle position determines the direction and region of the protein subjected to the force applied through the optical tweezers (i.e., pulling geometry) [15][16][17] . We generated three PKA regulatory subunit constructs with unique pulling geometries to probe cAMP binding coupled to inter-domain interactions (Fig 1b, right).…”