Computer simulations of ribosome reactions

Peptide bond formation and translational termination on the ribosome have been simulated by molecular mechanics, free energy perturbation, empirical valence bond (MD/FEP/EVB) and automated docking methods. Recent X-ray crystallographic data is used here to calculate the entire free energy surface for the system complete with substrates, ribosomal groups, solvent molecules and ions. A reaction mechanism for peptide bond formation emerges that is found to be catalyzed by the ribosome, in agreement with kinetic data and activation entropy measurements. The results show a water mediated network of hydrogen bonds, capable of reducing the reorganization energy during peptidyl transfer. The predicted hydrogen bonds and the structure of the active site were later confirmed by new X-ray structures with proper transition states analogs.Elongation termination on the ribosome is triggered by binding of a release factor (RF) protein followed by rapid release of the nascent peptide. The structure of the RF, bound to the ribosomal peptidyl transfer center (PTC), has not been resolved in atomic detail. Nor is the mechanism known, by which the hydrolysis proceeds. Using automated docking of a hepta-peptide RF fragment, containing the highly conserved GGQ motif, we identified a conformation capable of catalyzing peptide hydrolysis. The MD/FEP/EVB calculations also reproduce the slow spontaneous release when RF is absent, and rationalize available mutational data. The network of hydrogen bonds, the active site structure, and the reaction mechanism are found to be very similar for both peptidyl transfer and termination.New structural data, placing a ribosomal protein (L27) in the PTC, motivated additional MD/FEP/EVB simulations to determine the effect of this protein on peptidyl transfer…


1. Introduction
2. Computational methods used to describe chemical reactions
Quantum mechanics and electronic energy
Force fields
Molecular dynamics (MD)
Statistical thermodynamics and free energy
Free energy perturbation (FEP)
Empirical valence bond (EVB) method
Docking and scoring
3. Peptidyl transfer and chain termination on the ribosome
The ribosome
Functional states of the ribosome during protein synthesis
The structure of the peptidyl transfer center
4. Simulating peptidyl transfer on the ribosome
The catalytic effect of the ribosomal PTC
Predicting the structure of the PTC during peptidyl transfer
5. Simulation of structure and mechanism of elongation termination on the ribosome
Termination induced by deacylated A-site tRNA
Release factor induced termination in the PTC
6. L27, minimal substrates, and the pH-dependence of peptidyl transfer
Is the ribosome a ribozyme? – The ribosomal L27 protein in the PTC
The effect of Puromycin and L27 on pH-dependence
A-site substrates and the effect of mutations in the PTC
7. Summary in swedish–svensk sammanfattning
8. References

Author: Trobro, Stefan

Source: Uppsala University Library

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