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While extensive experimental data exist, studying real-time protein dynamics and enzyme catalysis remains challenging. Computational advances have been instrumental in overcoming experimental limitations, enabling molecular-level insights into biological macromolecules. The integration of experimental and computational approaches has proven to be very valuable in protein studies. Here, we demonstrate this synergy by investigating the conformational stability of the urethanase UMG-SP3, which exhibited a lower optimum temperature than expected and rapid loss of activity. Molecular dynamics simulations of the UMG-SP3-substrate complex at various temperatures revealed structural rearrangements outside the optimum temperature range (25\u201335\u202f\u00b0C), leading to loss of the native protein fold and impaired substrate binding. Even at the optimum temperature for activity, the enzyme struggled to maintain a catalytically favourable orientation, aligning with experimental findings. Unfolding profiles were determined through differential scanning fluorimetry. Notably, the computational results provided a rationaly for the structural instability observed experimentally, emphasizing the strength of computational methods in elucidating protein behaviour at the atomic level. This study highlights the importance of combining experimental and computational approaches to deepen our understanding of protein stability and function.<\/jats:p>","DOI":"10.1515\/pac-2025-0489","type":"journal-article","created":{"date-parts":[[2025,8,26]],"date-time":"2025-08-26T16:02:26Z","timestamp":1756224146000},"page":"1405-1418","source":"Crossref","is-referenced-by-count":0,"title":["Bridging experiment and theory: a computational exploration of UMG-SP3 dynamics"],"prefix":"10.1515","volume":"97","author":[{"given":"Lu\u00eds M. C.","family":"Teixeira","sequence":"first","affiliation":[{"name":"LAQV\/REQUIMTE, Departamento de Qu\u00edmica e Bioqu\u00edmica, Faculdade de Ci\u00eancias Universidade do Porto , Rua do Campo Alegre , s\/n, 4169-007 , Porto , Portugal"},{"name":"EnZync Center for Enzymatic Deconstruction of Thermoset Plastics, Aarhus , Denmark"}]},{"given":"Pedro","family":"Paiva","sequence":"additional","affiliation":[{"name":"LAQV\/REQUIMTE, Departamento de Qu\u00edmica e Bioqu\u00edmica, Faculdade de Ci\u00eancias Universidade do Porto , Rua do Campo Alegre , s\/n, 4169-007 , Porto , Portugal"},{"name":"EnZync Center for Enzymatic Deconstruction of Thermoset Plastics, Aarhus , Denmark"}]},{"given":"Pedro","family":"Ferreira","sequence":"additional","affiliation":[{"name":"LAQV\/REQUIMTE, Departamento de Qu\u00edmica e Bioqu\u00edmica, Faculdade de Ci\u00eancias Universidade do Porto , Rua do Campo Alegre , s\/n, 4169-007 , Porto , Portugal"},{"name":"EnZync Center for Enzymatic Deconstruction of Thermoset Plastics, Aarhus , Denmark"}]},{"given":"Laura","family":"Rotilio","sequence":"additional","affiliation":[{"name":"EnZync Center for Enzymatic Deconstruction of Thermoset Plastics, Aarhus , Denmark"},{"name":"Department of Biotechnology and Biomedicine , Technical University of Denmark , Lyngby , Denmark"}]},{"given":"Jens P.","family":"Morth","sequence":"additional","affiliation":[{"name":"EnZync Center for Enzymatic Deconstruction of Thermoset Plastics, Aarhus , Denmark"},{"name":"Department of Biotechnology and Biomedicine , Technical University of Denmark , Lyngby , Denmark"}]},{"given":"Daniel E.","family":"Otzen","sequence":"additional","affiliation":[{"name":"EnZync Center for Enzymatic Deconstruction of Thermoset Plastics, Aarhus , Denmark"},{"name":"Interdisciplinary Nanoscience Centre (iNANO) , Aarhus University , Gustav Wieds Vej 14, DK-8000 , Aarhus , Denmark"}]},{"given":"Pedro A.","family":"Fernandes","sequence":"additional","affiliation":[{"name":"LAQV\/REQUIMTE, Departamento de Qu\u00edmica e Bioqu\u00edmica, Faculdade de Ci\u00eancias Universidade do Porto , Rua do Campo Alegre , s\/n, 4169-007 , Porto , Portugal"},{"name":"EnZync Center for Enzymatic Deconstruction of Thermoset Plastics, Aarhus , Denmark"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9164-5271","authenticated-orcid":false,"given":"Maria J.","family":"Ramos","sequence":"additional","affiliation":[{"name":"LAQV\/REQUIMTE, Departamento de Qu\u00edmica e Bioqu\u00edmica, Faculdade de Ci\u00eancias Universidade do Porto , Rua do Campo Alegre , s\/n, 4169-007 , Porto , Portugal"},{"name":"EnZync Center for Enzymatic Deconstruction of Thermoset Plastics, Aarhus , Denmark"}]}],"member":"374","published-online":{"date-parts":[[2025,8,27]]},"reference":[{"key":"2025121816422071787_j_pac-2025-0489_ref_001","unstructured":"Bruce Alberts, A. 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