Julian O. Streit: The ribosome lowers the entropic penalty of protein folding

There is increasing evidence of a direct role for the ribosome in regulating folding of the nascent chain2,3,4,5,6,7,8,9,10, with increasing clarity on how it interacts with the elongating nascent chain7,11,12,13, which is thought to contribute to alterations to nascent chain thermodynamic stability6,7,8,9,10 and folding and unfolding rates5,8. Consequently, co-translational folding (coTF) differs from in vitro refolding studies of analogous, isolated counterparts3,5,6,14,15,16, with unique intermediate conformations in coTF3,5,6,14,15,17, folding in the absence of the complete protein sequence4,14, and the ability of the ribosome to mitigate misfolding-prone destabilizing mutations4 among the many discriminating observations whose origins remain poorly understood. 

This is a crucial gap in our understanding of proteostasis as many proteins reach an active conformation following coTF, whereas post-translational unfolding–refolding in the cell is generally avoided owing to high kinetic stabilities, and when proteins are unfolded (in vitro), they often do not refold spontaneously, but instead misfold and aggregate1,18,19.

In contrast to refolding studies, the unfolded state on the ribosome exists under native conditions7, and is adopted by all proteins during early biosynthesis. The ribosome-bound unfolded state has not been characterized in structural detail owing to technical challenges, yet is likely to be crucial to understanding folding thermodynamics and pathways20,21,22. Here, using paramagnetic relaxation enhancement (PRE) NMR spectroscopy (PRE-NMR) combined with atomistic molecular dynamics simulations, we have determined structural ensembles of the unfolded state and found that the ribosome structurally expands the conformational ensemble. We infer an entropically driven destabilization of the unfolded state on the ribosome relative to in isolation arising primarily from the increased solvation of the more expanded ensemble. Experiments show that this results in the ribosome reducing the entropic penalty of protein folding by up to around 30 kcal mol−1. 

Despite previous suggestions that interactions between nascent chains and the ribosome surface influence folding kinetics and thermodynamics5,6,7, we show here that these interactions account for a minor fraction of the energetic changes observed between protein folding on and off the ribosome. Instead, we establish that the entropic destabilization of the unfolded state provides the fundamental basis for why protein folding on the ribosome is distinct to refolding in vitro.

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