Automated Engineering Protein Dynamics via Loop Grafting: Improving <i>Renilla</i> Luciferase Catalysis
| Autoři | |
|---|---|
| Rok publikování | 2025 |
| Druh | Článek v odborném periodiku |
| Časopis / Zdroj | ACS Catalysis |
| Fakulta / Pracoviště MU | |
| Citace | |
| www | https://pubs.acs.org/doi/10.1021/acscatal.4c06207 |
| Doi | http://dx.doi.org/10.1021/acscatal.4c06207 |
| Klíčová slova | loop grafting; protein dynamics; bifunctionalprotein chimeras; Renilla luciferase; enzyme engineering; bioluminescent catalytic activity |
| Přiložené soubory | |
| Popis | Engineering protein dynamics is a challenging and unsolved problem in protein design. Loop transplantation or loop grafting has been previously employed to transfer dynamic properties between proteins. We recently released a LoopGrafter Web server to execute the loop grafting task, employing eight computational tools and one database. The LoopGrafter method relies on the prediction of the local dynamic behavior of the elements to be transplanted and has successfully reconstructed previously engineered sequences. However, it was unclear whether catalytically competitive previously uncharacterized designs could be obtained by this method. Here, we address this question, showing how LoopGrafter generates viable loop-grafted chimeras of luciferases, how these chimeras encompass the activity of interest and unique kinetic properties, and how all this process is done fully automatically and agnostic of any previous knowledge. All constructed designs proved to be catalytically active, and the most active one improved the activity of the template enzyme by 4 orders of magnitude. The computational details and parameter optimization of the sequence pairing step of the LoopGrafter workflow are revealed. The optimized and experimentally validated loop grafting workflow available as a fully automated Web server represents a powerful approach for engineering catalytically efficient enzymes by modification of protein dynamics. |
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