Computational Analysis of the Fully Activated Orexin Receptor 2 across Various Thermodynamic Ensembles with Surface Tension Monitoring and Markov State Modeling

In this study, we investigated the stability of the fully activated conformation of the orexin receptor 2 (OX₂R) embedded in a pure POPC bilayer using MD simulations. Various thermodynamic ensembles (i.e., <i>NPT</i>, <i>NVT</i>, <i>NVE</i>, <i>NPAT</i>, <i>μVT</i>, and <i>NPγT</i>) were employed to explore the dynamical heterogeneity of the system in a comprehensive way. In addition, informational similarity metrics (e.g., Jensen-Shannon divergence) as well as Markov state modeling approaches were utilized to elucidate the receptor kinetics. Special attention was paid to assessing surface tension within the simulation box, particularly under <i>NPγT</i> conditions, where 21 nominal surface tension constants were evaluated. Our findings suggest that traditional thermodynamic ensembles such as <i>NPT</i> 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 <i>NPγT</i> 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.