Chaperone-Guided Co-Translational Folding

Co-translational folding likely simplifies the conformational search problem for large proteins, but the events leading to correctly folded, functional structures remain poorly characterized. Domain-wise folding and help from chaperones are particularly important for multi-domain proteins, which constitute a large fraction of all proteomes. Using optical tweezers, we have dissected early folding events of nascent elongation factor G, a multi-domain protein that requires chaperones for folding. Interactions with the ribosome reduce inter-domain misfolding and, depending on nascent chain length, can either reduce or increase folding rates of the N-terminal G-domain. Successful completion of G-domain folding is crucial because it is a prerequisite for folding of the next domain. Unexpectedly, co-translational folding does not proceed unidirectionally: unfolded polypeptide emerging from the ribosome can denature an already folded domain. The chaperone trigger factor, but not the ribosome, protects against denaturation, thus helping multi-domain proteins overcome inherent challenges during co-translational folding. Misfolding among more C-terminal domains, which are energetically coupled in the native structure, poses an additional obstacle for EF-G folding. The second majoe nascent chain-binding chaperone in bacteria, DnaK, acts on these longer nascent chains and changes the folding energy landscape in fashion distinct from trigger factor. Our single-molecule experiments define the folding pathway of a complex multi-domain protein and show how the ribosome and two differentially acting chaperones together modulate nascent chain folding.