Biophysical Society Thematic Meeting| Lima 2019

Revisiting the Central Dogma of Molecular Biology at the Single-Molecule Level

Friday Speaker Abstracts

THE RIBOSOME COOPERATES WITH CHAPERONES TO GUIDE MULTIDOMAIN PROTEIN FOLDING

Christian M. Kaiser ; Kaixian Liu 1 ; Kevin Maciuba 1 ; 1 Johns Hopkins University, Biology, Baltimore, MD, USA

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 the complete folding pathway of elongation factor G, a multi-domain protein that requires chaperones for folding. Early during synthesis, 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 protects against denaturation, thus helping multi-domain proteins overcome inherent challenges during co-translational folding. In contrast, neither the ribosome nor the second major nascent chain-binding chaperone, the Hsp70 protein DnaK, prevent denaturation. Interestingly, we find that the energetic coupling among the three C-terminal domains prevents domain-wise folding from continuing. Instead, folding of domains III, IV and V can occur only synthesis is complete and the protein is released from the ribosome. DnaK binds to longer nascent chains and keeps them in a state competent for efficient post-translational folding. Our single-molecule experiments define the folding pathway of a complex multi-domain protein and demonstrate how the ribosome and two differentially acting chaperones together modulate nascent chain folding.

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