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. 2008 Nov 14;383(3):683-92.
doi: 10.1016/j.jmb.2008.07.035. Epub 2008 Jul 22.

Cotranslational folding promotes beta-helix formation and avoids aggregation in vivo

Affiliations

Cotranslational folding promotes beta-helix formation and avoids aggregation in vivo

Michael S Evans et al. J Mol Biol. .

Abstract

Newly synthesized proteins must form their native structures in the crowded environment of the cell, while avoiding non-native conformations that can lead to aggregation. Yet, remarkably little is known about the progressive folding of polypeptide chains during chain synthesis by the ribosome or of the influence of this folding environment on productive folding in vivo. P22 tailspike is a homotrimeric protein that is prone to aggregation via misfolding of its central beta-helix domain in vitro. We have produced stalled ribosome:tailspike nascent chain complexes of four fixed lengths in vivo, in order to assess cotranslational folding of newly synthesized tailspike chains as a function of chain length. Partially synthesized, ribosome-bound nascent tailspike chains populate stable conformations with some native-state structural features even prior to the appearance of the entire beta-helix domain, regardless of the presence of the chaperone trigger factor, yet these conformations are distinct from the conformations of released, refolded tailspike truncations. These results suggest that organization of the aggregation-prone beta-helix domain occurs cotranslationally, prior to chain release, to a conformation that is distinct from the accessible energy minimum conformation for the truncated free chain in solution.

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Figures

Figure 1
Figure 1. Tailspike structure and truncation constructs
Tailspike trimer crystal structure; one monomer chain is depicted as a blue ribbon and the other two chains are depicted as light and dark grey space filling models. mAb epitope boundaries are indicated by black circles. The portion of the tailspike polypeptide chain exposed outside the ribosome for each tailspike nascent chain construct is indicated by the red shading behind the crystal structure.
Figure 2
Figure 2. Binding of anti-tailspike mAbs to ribosome bound tailspike nascent chains and corresponding released chains
mAb binding affinity to ribosome bound tailspike nascent chains (solid bars), and mAb binding affinity to the either the corresponding released tailspike chain (for TSS, TMS and TβS), or native tailspike (for TFS) (open bars). ‘X’ indicates no measurable binding affinity was detected; ‘W’ indicates a weak affinity, too low to be quantified (Ka < 106 M−1); ‘B’ signifies high background binding of mAb 175 to other components in the cell lysate supernatant. Error bars represent the standard deviation between experiments performed in triplicate. mAbs to the right of the vertical dashed lines have epitopes not yet synthesized at these nascent chain lengths. Results for mAb 236 binding to TFS, TβS and TSS nascent chains were previously reported.
Figure 3
Figure 3. Protease digestion of ribosome-bound tailspike nascent chains
(a) Ribosomes bearing TFS nascent chains were treated with 1 μg/mL proteinase K for the indicated times at 4°C. The resulting fragments were separated on 5–14% denaturing polyacrylamide gels, transferred to a PVDF membrane, and immunoblotted using mAbs 70 and 92. (b) Ribosomes bearing TFS, TβS, TMS or TSS nascent chains were incubated for 5 minutes at 4°C with or without 1 μg/mL proteinase K. Digestion products were detected as in (a). Similar results were observed for room-temperature reactions, although the reactions proceeded more rapidly (data not shown).
Figure 4
Figure 4. Conformational differences between refolded Tβ and the corresponding nascent, or released, polypeptide chain
(a) mAb binding affinity to TβS released chains (open bars), and Tβ refolded, truncated protein (shaded bars). An ‘X’ indicates that no measurable binding affinity could be detected, and a ‘B’ signifies high background binding (see Figure 2). Error is reported as in Figure 2. (b) Proteolysis of refolded Tβ. Refolded Tβ was diluted to a concentration similar to that of ribosome-bound TβS in a solution containing ribosomes expressing an unrelated stalled nascent chain (cG49). Digestion of ribosome-bound TβS is shown for comparison. The proteolysis reactions were performed at 4°C and were visualized by western blotting as described in Figure 3.
Figure 5
Figure 5. Chaperone association with ribosome-bound tailspike nascent chains
(a) Cell lysates were centrifuged through sucrose cushions. The unpelleted lysate supernatant was removed, and the ribosomes were resuspended. Aliquots of both the unpelleted lysate and the ribosomes were examined by western blotting using anti-DnaK (top) or anti TF (bottom) antibodies. V, empty vector control; F, TFS; β, TβS; M, TMS; S, TSS. For DnaK, a vector control sample spiked with DnaK (1:1 DnaK:ribosome) is included as a positive control (V+D). (b) Hydropathy plot of tailspike. Mean hydropathy of the tailspike sequence was calculated according to published procedures. Putative TF binding sites, as indicated by a mean hydropathy of less than −0.5 kcal/mol, are shown in green. (c) Proteinase K digestion of TβS RNCs from MC4100 or MC4100Δtig cells. Proteolysis reactions were preformed at 4°C and visualized by western blotting as described in Figure 3.
Figure 6
Figure 6. Model for co-translational folding of tailspike nascent chains
When the first rungs of the β-helix emerge from the ribosome exit tunnel (TSS), little native-like or compact structure can form. As more of the β-helix is translated, a relatively stable compact structure containing native-like interactions forms throughout the first seven rungs of the β-helix domain (TMS). Once the entire β-helix domain is exposed from the ribosome (TβS), native-like compact structure forms throughout a significant portion of the chain, including the mAb 51 epitope (around residue 406). Based on the results of the protease protection experiments, the tailspike C-terminal interdigitated domain may be in a less compact conformation than the C-terminus of the β-helix domain. Free from the ribosome, truncated chains tend to aggregate, but the fraction remaining in solution maintains the conformation adopted while ribosome-bound. Chemical denaturation and refolding yields primarily insoluble aggregates, but the small amount of Tβ that refolds to a soluble, compact structure adopts a conformation that is distinct from the conformation of TβS while it is ribosome-bound.

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