Computational Analysis of Fully Activated Orexin Receptor 2 Across Various Thermodynamic Ensembles with Surface Tension Monitoring

Molecular dynamics (MD) simulations play a crucial role in understanding dynamic biological processes at nanoscale, yet predicting non-equilibrium phenomena like receptor activation presents significant challenges. In such cases, the primary objective isn't merely achieving stable MD trajectories; rather, it's imperative to remove all artificial restraints in order to unveil suppressed mechanical modes within the simulated systems, and thus advancing computer-aided drug design. In this study, we investigated the stability of the fully activated conformation of the orexin receptor 2 (OX2R) embedded in a pure 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) bilayer using MD simulations. Various thermodynamic ensembles (i.e. NPT, NVT, NVE, NPAT, µVT and NPγT) were employed to explore the system's dynamics comprehensively. 11 physical quantities, including so called OX2R activation distance, essential protein dynamics, membrane thickness, hydrogen order parameters, and ligand-protein potential energies, across 104 MD trajectories covering 10.4 µs of chemical time were calculated and profoundly analyzed. Special attention was given to assessing surface tension within the simulation box, particularly under NPγT conditions, where 21 nominal surface tension constants were evaluated. Notably, our findings suggest that traditional thermodynamic ensembles like NPT may not adequately control physical properties of the POPC membrane, impacting the plausibility of the OX2R model. In general, the performed study underscores the importance of employing the NPγT ensemble for computational investigations of membrane-embedded receptors, as it effectively maintains zero surface tension in the simulated system. These results offer valuable insights for future research aimed at understanding receptor dynamics and designing targeted therapeutics.