The ribosome destabilizes native and non‐native structures in a nascent multidomain protein

Abstract

Correct folding is a prerequisite for the biological activity of most proteins. Folding has largely been studied using in vitro refolding assays with isolated small, robustly folding proteins. A substantial fraction of all cellular proteomes is composed of multidomain proteins that are often not amenable to this approach, and their folding remains poorly understood. These large proteins likely begin to fold during their synthesis by the ribosome, a large molecular machine that translates the genetic code. The ribosome affects how folding proceeds, but the underlying mechanisms remain largely obscure. We have utilized optical tweezers to study the folding of elongation factor G, a multidomain protein composed of five domains. We find that interactions among unfolded domains interfere with productive folding in the full-length protein. The N-terminal G-domain constitutes an independently folding unit that, upon in vitro refolding, adopts two similar states that correspond to the natively folded and a non-native, possibly misfolded structure. The ribosome destabilizes both of these states, suggesting a mechanism by which terminal misfolding into highly stable, non-native structures is avoided. The ribosome may thus directly contribute to efficient folding by modulating the folding of nascent multidomain proteins.

Force-profile analysis of the cotranslational folding of HemK and filamin domains: Comparison of biochemical and biophysical folding assays

“…This tether length is clearly longer than what is required for folding of a similar-sized Ig-like domain such as the I27 titin domain that folds at a tether length of ~35 residues, as determined by FPA and cryo-EM [3]. The simplest explanation for the long tether length required for cotranslational folding of FLN5 is that the ribosome surface destabilizes the folded state relative to the unfolded state [14], as has been observed for other proteins when tethered to a ribosome [20,35,36].…”

A small single-domain protein folds through the same pathway on and off the ribosome

“…For multidomain proteins, the vectorial nature of cotranslational folding can affect the pathway by altering which domain folds first (15,29,30). Other studies have suggested the ribosome and/or translation may alter the stable intermediates that single domains sample during folding (16,23). Here, we focus on how attachment to the ribosome changes the folding barrier (i.e., the transition state) of a single protein domain that folds without stable intermediates.…”

“…Recent experiments harnessing the ability of the force generated by folding to release stalled nascent chains provide insight into the point during translation when different types of proteins fold (17)(18)(19)22). FRET and force spectroscopy experiments reveal the partially folded conformations some domains access before their entire sequence has left the exit tunnel (16,23). Pulse proteolysis and optical trapping experiments have been used to explore the energy landscape of RNCs outside the exit tunnel, showing that the ribosome significantly alters the stability and dynamics of proteins (13,14,23).…”

“…FRET and force spectroscopy experiments reveal the partially folded conformations some domains access before their entire sequence has left the exit tunnel (16,23). Pulse proteolysis and optical trapping experiments have been used to explore the energy landscape of RNCs outside the exit tunnel, showing that the ribosome significantly alters the stability and dynamics of proteins (13,14,23). Moreover, translation rates, which can be modulated by codon usage and mRNA secondary structure, affect the folding efficiency of a protein (24)(25)(26)(27)(28).…”

Mechanical Forces and Their Effect on the Ribosome and Protein Translation Machinery

“…Chaperones play an even more prominent role in the assembly of large multi-domain proteins, which are more prone to inter-domain misfolding events [48][49][50]. A recent study by Liu et al used optical tweezers to better to evaluate the synthesis and early folding intermediates of the multi-domain protein, EF-G, an essential mediator of ribosome mRNA translocation (as detailed in Section 1 and 2a) [49]. This study demonstrated that both the ribosome and TF help reduce any inter-domain misfolding so that the N-terminal G domain can fold efficiently.…”