The Ribosome and the Translocon
Modeling the ribosome and translocon systems present a major challenge for multilevel approaches.
Our studies of the ribosome started with the elucidation of the origin of teh catalytic power of the ribosome1
and understanding the activation of EF-Tu by the ribosome2,3
. Much more research is now in progress.
Another related problem that was studies by our group is the translocon and its complex with the ribosome.
The translocon is responsible for protein translocation across the membrane as well as their proper integration into the membrane through the so called lateral gate.
In exploring this system we investigated several puzzling questions related to the translocon-assisted membrane protein integration.
One of such questions involves the mechanism of membrane insertion of charged residues.
The discrepancy between the experimental and theoretical studies led to multiple attempts to resolve the issue.
To advance on this front we used our CG model to estimate the energetics of the transmembrane helix with central ionized arginine in the presence of the translocon and other helixes.
Our study indicated that the free energy of inserting arginine from water to membrane could be substantially reduced by interaction with other helix.
Another important question related to the translocon-assisted protein insertion is the mechanism of establishing proper membrane protein topology.
It is known from experiments that some mutations of the translocon or flanking residues of the peptide transmembrane domain affect final protein orientation in the membrane.
This subject is complicated by the fact that little is known about the mechanism of membrane integration as well as about the intermediate structures of the peptide during the insertion.
Here we challenged ourselves to obtain the complete free energy profile for the protein translocation through the translocon as well as membrane integration.
By applying several constraints on the system we were able to obtain the profile4
, which we used to investigate the effect of different mutations and the ribosome binding.
Comparison with experimental data led to the conclusion that the insertion process is most likely a non-equilibrium process and the peptide topology is controlled by the barrier of inserting into the translocon.
The obtained free energy profile allowed us to approach fundamental questions regarding the nature of the coupling between two large biological systems – translocon and ribosome.
That is, we investigated the origin of the biphasic pulling force from the translocon that, as was shown experimentally, allows releasing the stalling of the elongated nascent peptide chain from the ribosome.
Combining the estimate of the chemical barriers of the peptide bond formation for the regular and stalled peptide sequences with the profile for the translocon-assisted protein membrane integration and performing the Langevin dynamics simulations of the ribosome/translocon model, we were able to reproduce the experimental effect.
This and other studies highlight the importance of obtaining the free energy profile for the thorough understanding of the mechanisms underlying different biological processes.
The results of LD simulation of the peptide penetration process and the stalling effect.
The figure describes the time dependence of xistall and x1 for a peptide chain with 40 and 36 units, which corresponds to L = 31 (blue) and 27 (red), respectively. [The barriers used for the LD simulations were obtained by scaling down the energy terms by 0.43. This allowed simulating the insertion process in a relatively short time and then estimating the relevant time for the actual barriers by using the corresponding Boltzmann probability.]. The snapshots on the top and bottom of the plot shows the configuration of the nascent peptide chain for L = 31 and L = 27, respectively.
The ribosome and TR are shown schematically, the starting configuration of the nascent chain is in cyan, the leading particle (x1) is in red, and all other particles added to the growing chain are shown in magenta. The interpolated time (that should be obtained without scaling) for L = 31 and L = 27 are 6 min and 36 min, respectively. Other parameters can lead to a larger time difference.