Probing Protein-Ligand Unbinding Kinetics with Temperature-Coupled Molecular Dynamics

Traditional receptor–ligand theory prioritizes equilibrium affinity, but kinetic factors—especially the energy barriers for association and dissociation—are often important. Here, we hypothesize that protein–ligand dissociation proceeds via a well-defined activation barrier that can be quantified through physically realistic simulations. We employ a temperature-coupled molecular dynamics (TCMD) protocol that selectively heats the ligand while maintaining the protein near physiological temperature, thereby promoting unbiased dissociation events without sacrificing structural integrity. The resulting temperature-dependent off-rate follows a theoretically consistent trend, allowing robust extraction of activation energies. Applied to streptavidin–biotin benchmarks, TCMD reproduces experimental dissociation activation barriers with a mean absolute error of 1.2 kcal/mol. Beyond numerical agreement, TCMD provides high-resolution mechanistic insight by showing how interaction-energy fluctuations, hydrogen-bond reordering, and discrete conformational changes shape the exit pathway, thereby linking the estimated barrier to its structural origins. These results establish, with near-experimental fidelity, the activation barrier governing dissociation, encouraging a move beyond affinity-only perspectives. Furthermore, the unbiased trajectories provide a detailed, time-resolved view of contact reorganization during dissociation, offering a realistic framework for studying binding kinetics at atomic resolution.