Molecular dynamics simulations of drug binding frequently assign fixed protonation states even though the measured affinity reflects an ensemble of ligand, receptor, and complex states under defined experimental conditions. This discrepancy can complicate free-energy interpretation, particularly when proton exchange is coupled to hydration, electrostatic polarization, or conformational rearrangement. This theoretical study develops a mechanism-based account of how microscopic protonation equilibria contribute to observed binding behavior and why ostensibly protonation-aware calculations may still disagree with assay measurements. Recent evidence from multistate free-energy methods, constant-pH molecular dynamics, ionization prediction, and protein–ligand investigations is integrated with a critical experimental–computational counterexample. The analysis separates chemical-state populations, state-conditioned association, and assay-level observability, while retaining alternative explanations involving solvent, force fields, and molecular accessibility. It proposes that concordance requires explicit specification of the states and thermodynamic conditions to which each calculated or measured quantity refers. Discriminating comparisons are identified for evaluating proton linkage against competing mechanisms. The account is nonempirical and does not establish a universally predictive method; its contribution is a falsifiable interpretation of when molecular simulations and binding assays can legitimately be compared.
IJPRAS
