Molecular simulations of cotranslational protein folding: fragment stabilities, folding cooperativity and trapping in the ribosome tunnel

Abstract

Although molecular simulation methods have yielded valuable insights into mechanistic aspects of protein refolding in vitro, they have up to now not been used to model the folding of proteins as they are actually synthesized by the ribosome. To address this issue, we report here simulation studies of three model proteins: chymotrypsin inhibitor 2 (CI2), barnase, and Semliki forest virus protein (SFVP), and directly compare their folding during ribosome-mediated synthesis with their refolding from random, denatured conformations. To calibrate the methodology, simulations are first compared with in vitro data on the folding stabilities of N-terminal fragments of CI2 and barnase; the simulations reproduce the fact that both the stability and thermal folding cooperativity increase as fragments increase in length. Coupled simulations of synthesis and folding for the same two proteins are then described, showing that both fold essentially post-translationally, with mechanisms effectively identical to those for refolding. In both cases, confinement of the nascent polypeptide chain within the ribosome tunnel does not appear to promote significant formation of native structure during synthesis; there are however clear indications that the formation of structure within the nascent chain is sensitive to location within the ribosome tunnel, being subject to both gain and loss as the chain lengthens. Interestingly, simulations in which CI2 is artificially stabilized show a pronounced tendency to become trapped within the tunnel in partially folded conformations: non-cooperative folding, therefore, appears in the simulations to exert a detrimental effect on the rate at which fully folded conformations are formed. Finally, simulations of the two-domain protease module of SFVP, which experimentally folds cotranslationally, indicate that for multi-domain proteins, ribosome-mediated folding may follow different pathways from those taken during refolding. Taken together, these studies provide a first step toward developing more realistic methods for simulating protein folding as it occurs in vivo.

Lattice simulations of cotranslational folding of single domain proteins

“…The simulation results reported here can be reconciled with available experimental data, but their full validation will depend on forthcoming CF experiments. Future computer simulations will use more detailed coarse grained models, which will provide protein specific predictions 14…”

“…This result was attributed to the fact that CF facilitates the assembly of local native structure that in turn steers folding away from the local energy minima, which act as RF kinetic traps. The computational study using Go off‐lattice models performed by Elcock14 compared the folding during ribosome‐mediated synthesis with refolding from random denatured conformations for several proteins, including chymotrypsin inhibitor 2 (CI2) and barnase. Those simulations suggested that both CI2 and barnase fold post‐translationally via a mechanism identical to that of RF.…”

Probing ribosome-nascent chain complexes produced in vivo by NMR spectroscopy

“…Its dimensions [Ϸ80-Å long and 10-to 20-Å wide at its narrowest point (20)], together with its conservative global flexibility (21), suggests that complete NC folding can only take place once it has emerged from the tunnel (22).…”

Protein escape at the ribosomal exit tunnel: Effect of the tunnel shape

“…This can be understood as due to two reasons: the faster diffusion at this temperature helps the protein to avoid trapped conformations, and the larger thermal fluctuations help the protein to get out from the traps. The trapping at an atomistic ribosome tunnel and the alleviation of trapping by increased thermal fluctuations have been also observed for the chymotrypsin inhibitor 2 (CI2) protein in an early simulation study by Elcock 14 . Our results here for GB1 are consistent with that previous work.…”

“…The latter can be observed near the tunnel exit port where there is enough space to hold the structure. Simulations 14,15 and experiments [16][17][18] indicate that cotranslational folding starts inside the tunnel, with the structures ranging from a non-native compact conformation 16 and transient tertiary structures 15 to a small protein domain 17 . There are also considerations that the folding inside the ribosome tunnel is negligible leading to a focus only on the folding of nascent chains as they emerge from the tunnel, as shown in studies with stalled ribosome-bound nascent chain experiments 19,20 and simulations 21,22 .…”