Gradual Compaction of the Nascent Peptide During Cotranslational Folding on the Ribosome

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>Nascent polypeptides begin to fold in the constrained space of the ribosomal peptide exit tunnel. Here we use force profile analysis (FPA) and photo-induced energy-transfer fluorescence correlation spectroscopy (PET-FCS) to show how a small α-helical domain, the N-terminal domain of HemK, folds cotranslationally. Compaction starts vectorially as soon as the first α-helical segments are synthesized. As nascent chain grows, emerging helical segments dock onto each other and continue to rearrange at the vicinity of the ribosome. Inside or in the proximity of the ribosome, the nascent peptide undergoes structural fluctuations on the μs time scale. The fluctuations slow down as the domain moves away from the ribosome. Folding mutations have little effect on folding within the exit tunnel, but abolish the final domain stabilization. The results show the power of FPA and PET-FCS in solving the trajectory of cotranslational protein folding and in characterizing the dynamic properties of folding intermediates.</jats:p>

Co‐translational folding of nascent polypeptides: Multi‐layered mechanisms for the efficient biogenesis of functional proteins

“…An implication of such a "co-translational folding force" is that it is a universal consequence when the nascent chain forms a stable globular structure close to the ribosome. Indeed, this effect has successfully been exploited to follow nascent chain folding with force-sensing elongation arrest peptides in vivo [47,53,54] and in vitro [44,48,55,56] (Figure 3).…”

Mechanisms of Cotranslational Protein Maturation in Bacteria

“…Going beyond the formation of an alpha-helical secondary structure, some small domains can also fold within the vestibule. This includes nascent chain compaction and the formation of beta-hairpins (Kosolapov and Deutsch, 2009;O'brien et al, 2010;Tu et al, 2014), as well as native folding of the zinc finger domain of ADR1 (Nilsson et al, 2015) or folding of the N-terminal domain of HemK inside the ribosomal exit tunnel (Liutkute et al, 2020a). However, the prevalence of such folding events in the tunnel is not yet clear.…”

“…As the nascent chain emerges from the ribosome, the spatial constraints of the tunnel are relieved while the limiting impact of the ribosome on the conformational space of the nascent chain partially remains. Supported by studies on multiple model proteins (Hsu et al, 2007;Ellis et al, 2008;Ellis et al, 2009;Kelkar et al, 2012;Holtkamp et al, 2015;Kim et al, 2015;Koubek et al, 2017;Nilsson et al, 2017;Farias-Rico et al, 2018;Mercier and Rodnina, 2018;Kemp et al, 2019;Liutkute et al, 2020a) it is estimated that at least 30% of the cytosolic E. coli proteome folds independently of chaperones (Ciryam et al, 2013). Folding of these proteins is therefore solely determined by the intrinsic biophysical properties of the amino acid sequence and the influence of the ribosome.…”

“…Upon emergence from the ribosome, the N-terminal domain of nascent HemK acquires an intermediate folding state within the ribosomal exit tunnel and rapidly folds into a native-like structure once the full domain has emerged. In solution, however, the HemK N-domain undergoes rapid transitions between folded and unfolded states without stable folding intermediates (Holtkamp et al, 2015;Mercier and Rodnina, 2018;Nissley and O'Brien, 2018;Kemp et al, 2019;Liutkute et al, 2020a). Similarly, the cytoskeletal protein spectrin was suggested to have differing folding pathways on and off the ribosome (Nilsson et al, 2017;Kemp et al, 2020).…”

Cotranslational folding of a periplasmic protein domain inEscherichia coli

“…In contrast, the cotranslational folding of cytosolic proteins can be studied in considerable detail, both in vitro and in vivo , using FRET-based assays [6, 7], protease-protection assays [6, 8–13], NMR [14–19], cryo-electron microscopy [20–22], optical tweezer experiments [23–26], and force-profile analysis (FPA) [7, 13, 20–22, 26–31]. These methods can track where in the ribosome exit tunnel a protein starts to fold, whether any folding intermediates form before the nascent protein has been fully synthesized, and to what extent the presence of the ribosome affects the folding kinetics and the thermodynamic stability of the folded state.…”