Protein evolution and protein engineering techniques are of great interest in basic science and industrial applications such as pharmacology, medicine, or biotechnology. Ancestral sequence reconstruction (ASR) is a powerful technique for probing evolutionary relationships and engineering robust proteins with good thermostability and broad substrate specificity. The following protocol describes the setting up and execution of an automated FireProtASR workflow using a dedicated web site. The service allows for inference of ancestral proteins automatically, from a single protein sequence. Once a protein sequence is submitted, the server will build a dataset of homology sequences, perform a multiple sequence alignment (MSA), build a phylogenetic tree, and reconstruct ancestral nodes. The protocol is also highly flexible and allows for multiple forms of input, advanced settings, and the ability to start jobs from: (i) a single sequence, (ii) a set of homologous sequences, (iii) an MSA, and (iv) a phylogenetic tree. This approach automates all necessary steps and offers a way for novices with limited exposure to ASR techniques to improve the properties of a protein of interest. The technique can even be used to introduce catalytic promiscuity into an enzyme. A web server for accessing the fully automated workflow is freely accessible at https://loschmidt.chemi.muni.cz/fireprotasr/. © 2021 Wiley Periodicals LLC. Basic Protocol: ASR using the Web Server FireProtASR.

Department of Information Systems Faculty of Information Technology Brno University of Technology Brno Czech Republic

International Clinical Research Center St Anne's University Hospital Brno Brno Czech Republic

Loschmidt Laboratories Department of Experimental Biology and RECETOX Faculty of Science Masaryk University Brno Czech Republic

Curr Protoc. 2022 Aug;2(8):e552. doi: 10.1002/cpz1.552. PubMed

Curr Protoc. 2022 Aug;2(8):e551. doi: 10.1002/cpz1.551. PubMed

Zobrazit více v PubMed

Ashkenazy, H., Penn, O., Doron-Faigenboim, A., Cohen, O., Cannarozzi, G., Zomer, O., & Pupko, T. (2012). FastML: A web server for probabilistic reconstruction of ancestral sequences. Nucleic Acids Research, 40(W1), W580-W584. doi: 10.1093/nar/gks498.

Babkova, P., Dunajova, Z., Chaloupkova, R., Damborsky, J., Bednar, D., & Marek, M. (2020). Structures of hyperstable ancestral haloalkane dehalogenases show restricted conformational dynamics. Computational and Structural Biotechnology Journal, 18, 1497-1508. doi: 10.1016/j.csbj.2020.06.021.

Babkova, P., Sebestova, E., Brezovsky, J., Chaloupkova, R., & Damborsky, J. (2017). Ancestral haloalkane dehalogenases show robustness and unique substrate specificity. ChemBioChem, 18(14), 1448-1456. doi: 10.1002/cbic.201700197.

Boutet, E., Lieberherr, D., Tognolli, M., Schneider, M., Bansal, P., Bridge, A. J., … Xenarios, I. (2016). UniProtKB/Swiss-Prot, the manually annotated section of the UniProt KnowledgeBase: How to use the entry view. In D. Edwards (Ed.), Plant bioinformatics, (pp. 23-54). New York, NY: Humana Press.

Chaloupkova, R., Liskova, V., Toul, M., Markova, K., Sebestova, E., Hernychova, L., … Damborsky, J. (2019). Light-emitting dehalogenases: Reconstruction of multifunctional biocatalysts. ACS Catalysis, 9, 4810-4823. doi: 10.1021/acscatal.9b01031.

Charleston, M. (2013). Phylogeny. S. Maloy and K. Hughes (Eds.), Brenner's encyclopedia of genetics, (pp. 324-325).

Diallo, A. B., Makarenkov, V., & Blanchette, M. (2010). Ancestors 1.0: A web server for ancestral sequence reconstruction. Bioinformatics, 26(1), 130-131. doi: 10.1093/bioinformatics/btp600.

Foley, G., Mora, A., Ross, C. M., Bottoms, S., Sützl, L., Lamprecht, M. L., … Bodén, M. (2019). Identifying and engineering ancient variants of enzymes using Graphical Representation of Ancestral Sequence Predictions (GRASP). BioRxiv, 2019-2012. doi: 10.1101/2019.12.30.891457.

Gaucher, E. A. (2007). Ancestral sequence reconstruction as a tool to understand natural history and guide synthetic biology: Realizing and extending the vision of Zuckerkandl and Pauling. In D. A. Liberles (Ed.), Ancestral sequence reconstruction, (pp. 20-33). Oxford, UK: Oxford University Press.

Gaucher, E. A., Govindarajan, S., & Ganesh, O. K. (2008). Palaeotemperature trend for Precambrian life inferred from resurrected proteins. Nature, 451(7179), 704-707. doi: 10.1038/nature06510.

Hanson-Smith, V., & Johnson, A. (2016). PhyloBot: A web portal for automated phylogenetics, ancestral sequence reconstruction, and exploration of mutational trajectories. PLoS Computational Biology, 12(7), e1004976. doi: 10.1371/journal.pcbi.1004976.

Letunic, I., & Bork, P. (2019). Interactive Tree Of Life (iTOL) v4: Recent updates and new developments. Nucleic Acids Research, 47(W1), W256-W259. doi: 10.1093/nar/gkz239.

Liberles, D. A. (Ed.). (2007). Ancestral sequence reconstruction. Oxford, UK: Oxford University Pres.

Matasci, N., & McKay, S. (2013). Phylogenetic analysis with the iPlant discovery environment. Current Protocols in Bioinformatics, 42(1), 6-13. doi: 10.1002/0471250953.bi0613s42.

Olsen, G. (1990). The” Newick's 8: 45” tree format standard. Available at http://evolution.genetics.washington.edu/phylip/newick_doc.html.

Phylogeny. (2013). 324-325.

Procter, J. B., Thompson, J., Letunic, I., Creevey, C., Jossinet, F., & Barton, G. J. (2010). Visualization of multiple alignments, phylogenies and gene family evolution. Nature Methods, 7(3), S16-S25. doi: 10.1038/nmeth.1434.

Ribeiro, A. J. M., Holliday, G. L., Furnham, N., Tyzack, J. D., Ferris, K., & Thornton, J. M. (2018). Mechanism and Catalytic Site Atlas (M-CSA): A database of enzyme reaction mechanisms and active sites. Nucleic Acids Research, 46(D1), D618-D623. doi: 10.1093/nar/gkx1012.

Sievers, F., & Higgins, D. G. (2018). Clustal Omega for making accurate alignments of many protein sequences. Protein Science, 27(1), 135-145. doi: 10.1002/pro.3290.

Štěpánková, V. (2013). Expansion of access tunnels and active-site cavities influences activity of haloalkane dehalogenases in organic cosolvents. Haloalkane dehalogenases in non-conventional reaction media. ChemBioChem, 14(7), 79. doi: 10.1002/cbic.201200733.

Taylor, W. R. (1997). Residual colours: A proposal for aminochromography. Protein Engineering, 10(7), 743-746. doi: 10.1093/protein/10.7.743.

Watanabe, K., Ohkuri, T., Yokobori, S. I., & Yamagishi, A. (2006). Designing thermostable proteins: Ancestral mutants of 3-isopropylmalate dehydrogenase designed by using a phylogenetic tree. Journal of Molecular Biology, 355(4), 664-674. doi: 10.1016/j.jmb.2005.10.011.

Waterhouse, A., Bertoni, M., Bienert, S., Studer, G., Tauriello, G., Gumienny, R., … Lepore, R. (2018). SWISS-MODEL: Homology modelling of protein structures and complexes. Nucleic Acids Research, 46(W1), W296-W303. doi: 10.1093/nar/gky427.

Wheeler, L. C., Lim, S. A., Marqusee, S., & Harms, M. J. (2016). The thermostability and specificity of ancient proteins. Current Opinion in Structural Biology, 38, 37-43. doi: 10.1016/j.sbi.2016.05.015.