Probing the mechanisms of unnatural protein translation with an engineered genetic code and orthogonal translational pairs
Final Report Abstract
The main goal of the proposal was to rationalize the effects leading to the ribosome stalling on oligoproline stretches in translation. First, we devised a few studies with amino acids models, in order to characterize different aspect of proline chemistry, with the specific aim to generate parameters for characterization of folding bias and chemical reactivity. This was done through the help of a set of a chemical analogues of proline. This design helped us to exclude the chemical reactivity of the main chain groups as the main cause for the translation arrest of the ribosome, as this was evident from model chemical experiments in solution. Furthermore, a devised set of proline analogues was supplied to collaboration partners within FOR 1805 in Daniel Wilson, Marina Rodnina and Ingo Wohlgemuth labs. Their examinations also showed absence of the chemical reactivity correlation with the speed or translation and efficiency of the stalling phenomenon. Our partners discovered that the rate of the peptidyl transfer acceleration by elongation factor P was nearly same for the proline analogues, although their starting translation rates were drastically different. Then they also found that the acceleration of the reaction is a result of an entropic effect coming from the elongation factor P binding to the ribosome. Second, to further understand this entropic effect, we performed molecular dynamic simulations. This part of the project was done in close collaboration with Wilson, Rodnina, Beckmann and Grubmüller groups, who supplied this study with the cryo-EM data. The molecular dynamics simulations demonstrated that binding of elongation factor P restricts conformational states of the tRNA positioned at the P-site of the ribosome, thereby changing the conformational landscape and leading to the preorganization effect for the peptidyl transfer. In this way, we fully explained the entropic effect from elongation factor P. The chemical structure of the elongation factor P, which leads to this effect includes essential post-translational modification at lysine 34. This side chain extends to the peptidyl transfer center when the protein is bond to the ribosome. To further address chemical variability of this structure, we designed a part of the project, where we included different chemical analogues at position 34 of the protein through the help of orthogonal translation. Only a few preliminary experiments were done in this direction due to cuts in funding. This issue remains unresolved to date, neither from our study nor in the literature. The orthogonal translation issues was the second major direction of this project. The incorporation of an arbitrary amino acid in place of a stop codon (usually amber) is a long term project in our laboratory and others. This technology is still far from being scaled up for massive indusrtical explorations, despite its proven efficiency on analytical scale. In collaboration with Ignatova lab and external partners we examined the efficiency of orthogonal translation in the presence of in frame stop codon, addressed efficiency of the aminoacyl tRNA synthetase, mRNA context effects, competition with release factor. These efforts helped us (as well as other experts in the field), to optimize the mRNA sequences and strain configurations for better protein productions. We found that the toxic effects of the amino acids involved in metabolic production and amino acid charging should also be taken into account, when addressing translation process. As a perspective, the project resulted in the discovery of a hydrophobic and a transmembrane polyproline helices. The exploration of these new-to-nature motifs, their structure assembly (e.g. in collagen helix) and applications will be pursued in the frame of other projects in the forthcoming future.
Publications
- Entropic contribution of elongation factor P to proline positioning at the catalytic center of the ribosome. J. Am. Chem. Soc. 137, 12997-13006 (2015)
L. K. Doerfel, I. Wohlgemuth, V. Kubyshkin, A. L. Starosta, D. N. Wilson, N. Budisa, M. V. Rodnina
(See online at https://doi.org/10.1021/jacs.5b07427) - A combined cryo-EM and molecular dynamics approach reveals the mechanism of ErmBL- mediated translation arrest. Nat Commun. 7, 12026 (2016)
Arenz S, Bock L V., Graf M, Innis CA, Beckmann R, Grubmüller H, Vaiana AC, Wilson DN
(See online at https://doi.org/10.1038/ncomms12026) - Discharging tRNAs: a tug of war between translation and detoxification in Escherichia coli. Nucleic Acids Res. 44, 8324-8334 (2016)
I. Avcilar-Kucukgoze, A. Bartholomäus, J. Cordero Varela, R. Kaml, P. Neubauer, N. Budisa, Z. Ignatova
(See online at https://doi.org/10.1093/nar/gkw697) - Energetic contribution to both acidity and conformational stability in peptide models. New J. Chem. 40, 5209-5220 (2016)
V. Kubyshkin, P. Durkin, N. Budisa
(See online at https://doi.org/10.1039/c5nj03611a) - Structural Basis for Polyproline-Mediated Ribosome Stalling and Rescue by the Translation Elongation Factor EF-P. Mol Cell. 68, 515-527 (2017)
Huter P, Arenz S, Bock L V., Graf M, Frister JO, Heuer A, Peil L, Starosta AL, Wohlgemuth I, Peske F, Nováček J, Berninghausen O, Grubmüller H, Tenson T, Beckmann R, Rodnina MV., Vaiana AC, Wilson DN
(See online at https://doi.org/10.1016/j.molcel.2017.10.014) - Transmembrane polyproline helix. J. Phys. Chem. Lett. 9, 2170-2174 (2018)
V. Kubyshkin, S. L. Grage, J. Bürck, A. S. Ulrich, N. Budisa
(See online at https://doi.org/10.1021/acs.jpclett.8b00829)
