Structural Origins of FRET-Observed Nascent Chain Compaction on the Ribosome

Abstract

A fluorescence signal arising from a Förster resonance energy transfer process was used to monitor conformational changes of a domain within the E. coli protein HemK during its synthesis by the ribosome. An increase in fluorescence was observed to begin 10 s after translation was initiated, indicating the domain became more compact in size. Since fluorescence only reports a single value at each time point it contains very little information about the structural ensemble that gives rise to it. Here, we supplement this experimental information with coarse-grained simulations that describe protein conformations and transitions at a spatial resolution of 3.8 Å. We use these simulations to test three hypotheses that might explain the cause of domain compaction: (1) that poor solvent quality conditions drive the unfolded state to compact, (2) that a change in the dimension of the space the domain occupies upon moving outside the exit tunnel causes compaction, or (3) that domain folding causes compaction. We find that domain folding and dimensional collapse are both consistent with the experimental data, while poor-solvent collapse is inconsistent. We identify alternative dye labeling positions on HemK that upon fluorescence can differentiate between the domain folding and dimensional collapse mechanisms. Partial folding of domains has been observed in C-terminally truncated forms of proteins. Therefore, it is likely that the experimentally observed compact state is a partially folded intermediate consisting, according to our simulations, of the first three helices of the HemK N-terminal domain adopting a native, tertiary configuration. With these simulations we also identify the possible cotranslational folding pathways of HemK.

Gradual compaction of the nascent peptide during cotranslational folding on the ribosome

“…However, the observation that proline substitutions, that are expected to alter helix formation, also reduce the tension suggest that the FPA high-tension regions reflect folding events inside the exit tunnel ( Figure 1d and Figure 1—figure supplement 1g ). Changes in folding trajectories upon perturbing protein structure were also demonstrated by FPA for other proteins ( Lu and Deutsch, 2005 ; Bhushan et al, 2010 ; Nilsson et al, 2015 ; Farías-Rico et al, 2018 ; Nissley and O'Brien, 2018 ). FPA studies also demonstrate that the growing nascent chain continues to undergo structural adjustments after emerging from the exit tunnel.…”

“…Upon continued synthesis, emerging helices begin to interact with one another forming tertiary intermediates inside the peptide exit tunnel ( Figure 1d ). The formation of individual α-helices and tertiary interactions between α-helical elements within the exit tunnel are well documented ( Lu and Deutsch, 2005 ; Bhushan et al, 2010 ; Nilsson et al, 2015 ; Farías-Rico et al, 2018 ; Nissley and O'Brien, 2018 ). Our results are consistent with the notion that folding of HemK NTD is sequential ( Mercier and Rodnina, 2018 ) and show several tension-generating steps corresponding to folding intermediates inside and outside of the ribosome ( Kemp et al, 2019 ).…”

Cotranslational Folding of Proteins on the Ribosome

“…By contrast, a polar nascent chain in this same situation would experience a smaller drive to form helical structures, but would rather displace the ordered solvent molecules on the surface of the tunnel resulting in close contact between the nascent chain and the tunnel walls [39]. Biochemical and structural studies suggest that nascent chains may form helical structures in the upper regions of the tunnel, even though a peptide with the same amino acid sequence in solution does not form a stable helix [37,40,41].…”

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

“…These include real-time FRET analysis [1], and methods in which nascent polypeptide chains of defined lengths are arrested in the ribosome and their folding status analyzed by, e.g., cryo-EM [2,3], protease resistance [1,[4][5][6][7][8][9], NMR [10][11][12][13][14][15], photoinduced electron transfer (PET) [1,16], folding-associated cotranslational sequencing [17], optical tweezer pulling [18][19][20][21], fluorescence measurements [22], and measuring the force that the folding protein exerts on the nascent chain using a translational arrest peptide (AP) as a force sensor [2,3,9,21,[23][24][25]. Further, coarse-grained molecular dynamics simulations of various flavors can provide detailed insights into cotranslational folding reactions [26][27][28][29][30], especially when coupled with experimental studies [3,26,31].…”