Molecular chaperones are central to protein homeostasis, guiding nascent polypeptides toward their native conformations and preventing misfolding and aggregation within the crowded cellular environment. Immediately post-translational events accompanying the release of newly synthesized chains from the ribosome lead to soluble native states and soluble and/or insoluble aggregates. Given that all three classes of species are typically kinetically trapped from each other after being initially generated, the early stages of protein life are crucial. Understanding how chaperones behave at this stage is essential to define the origin of folding versus aggregation pathways. Here, we focus on the ribosome-associated trigger factor (TF) chaperone, whose cotranslational interaction with nascent proteins at the ribosomal exit tunnel is poorly understood. Using a monomeric globin as a model system, we probe how TF interacts with the globin chain and how it influences its structure and dynamics during biosynthesis. This work synergistically employs cell-free transcription-translation, unnatural tRNA technologies, single-particle cryo-electron microscopy and fluorescence lifetime/anisotropy-decay spectroscopy to probe three-dimensional structure and dynamics of ribosome nascent-protein complexes. Taken together, our results reveal how TF prevents cotranslational aggregation while preserving nascent-chain conformational sampling. This research paves the way to a deeper molecular-level understanding of kinetic partitioning between protein folding and aggregation at birth.
Millan et al. (2026) studied this question.