The Ribosome Alters the Folding of a Multidomain Nascent Protein

Abstract

Protein folding is often studied in the context of full-length polypeptides in solution. However, the post-translational folding pathway may not recreate the pathway of folding when a protein is being synthesized by the ribosome. The effects of the ribosome and the rate of translation on folding are not well understood, partially due to the difficulty of probing the nascent chain without also affecting the ribosome. Using optical tweezers, we can look in detail at the nascent chain at the single-molecule level with minimal perturbation to the ribosome. Previous studies have shown that the ribosomal surface can act electrostatically to slow the kinetics of folding. Those studies used a linker to vary the distance from the surface and did not look at changes in sequence availability or multidomain proteins. To further understand cotranslational folding, we are studying the folding pathway of a two-domain calcium-binding protein calerythrin. Our results show that the full-length protein in solution folds robustly through a C-domain intermediate, but truncated versions of the protein can fold to an N domain or a misfolded state. During translation, the C-terminal residues required for native folding are not yet available, but the other observed intermediates could fold, in principle. By observing the folding at various sequence positions, and thus nascent chain lengths, we found that the ribosome can not only slow folding, but can also affect the unfolding rates, particularly increasing the unfolding rate of the misfolded state. The ribosome thus prevents the formation of this unproductive state via two mechanisms: decreasing the probability of folding and decreasing the stability of the folded state. This study gives further insight into the importance of the ribosome to regulate protein structure, and opens up new questions about the interplay between elongation and folding.