The quest to predict and understand protein evolution has been hindered by limitations on both the theoretical and the experimental fronts. Most existing theoretical models of evolution are descriptive, rather than predictive, leaving the final modifications in the hands of researchers. Existing experimental techniques to help probe the evolutionary sequence space of proteins, such as directed evolution, are resource-intensive and require specialised skills. We present the successor sequence predictor (SSP) as an innovative solution. Successor sequence predictor is an in silico protein design method that mimics laboratory-based protein evolution by reconstructing a protein's evolutionary history and suggesting future amino acid substitutions based on trends observed in that history through carefully selected physicochemical descriptors. This approach enhances specialised proteins by predicting mutations that improve desired properties, such as thermostability, activity, and solubility. Successor Sequence Predictor can thus be used as a general protein engineering tool to develop practically useful proteins. The code of the Successor Sequence Predictor is provided at https://github.com/loschmidt/successor-sequence-predictor , and the design of mutations will be also possible via an easy-to-use web server https://loschmidt.chemi.muni.cz/fireprotasr/ . SCIENTIFIC CONTRIBUTION: The Successor Sequence Predictor advances protein evolution prediction at the amino acid level by integrating ancestral sequence reconstruction with a novel in silico approach that models evolutionary trends through selected physicochemical descriptors. Unlike prior work, SSP can forecast future amino acid substitutions that enhance protein properties such as thermostability, activity, and solubility. This method reduces reliance on resource-intensive directed evolution techniques while providing a generalizable, predictive tool for protein engineering.

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

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