Bridging experiment and theory: a computational exploration of UMG-SP3 dynamics

Abstract Understanding how dynamical behaviour and structural features influence protein function and stability is crucial. 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 (25–35 °C), 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.