FireProtASR: A Web Server for Fully Automated Ancestral Sequence Reconstruction.

There is a great interest in increasing proteins' stability to widen their usability in numerous biomedical and biotechnological applications. However, native proteins cannot usually withstand the harsh industrial environment, since they are evolved to function under mild conditions. Ancestral sequence reconstruction is a well-established method for deducing the evolutionary history of genes. Besides its applicability to discover the most probable evolutionary ancestors of the modern proteins, ancestral sequence reconstruction has proven to be a useful approach for the design of highly stable proteins. Recently, several computational tools were developed, which make the ancestral reconstruction algorithms accessible to the community, while leaving the most crucial steps of the preparation of the input data on users' side. FireProtASR aims to overcome this obstacle by constructing a fully automated workflow, allowing even the unexperienced users to obtain ancestral sequences based on a sequence query as the only input. FireProtASR is complemented with an interactive, easy-to-use web interface and is freely available at https://loschmidt.chemi.muni.cz/fireprotasr/.

Fully Automated Ancestral Sequence Reconstruction using FireProtASR.

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.