A recent molecular dynamics investigation into the stability of hemoglobin concluded that the unliganded protein is only stable in the T state when a solvent box is used in the simulations that is ten times larger than what is usually employed (El Hage et al., 2018). Here, we express three main concerns about that study. In addition, we find that with an order of magnitude more statistics, the reported box size dependence is not reproducible. Overall, no significant effects on the kinetics or thermodynamics of conformational transitions were observed.

Recent molecular dynamics (MD) simulations of human hemoglobin (Hb) give results in disagreement with experiment. Although it is known that the unliganded (T${}_0$) and liganded (R${}_4$) tetramers are stable in solution, the published MD simulations of T${}_0$ undergo a rapid quaternary transition to an R-like structure. We show that T${}_0$ is stable only when the periodic solvent box contains ten times more water molecules than the standard size for such simulations. The results suggest that such a large box is required for the hydrophobic effect, which stabilizes the T${}_0$ tetramer, to be manifested. Even in the largest box, T${}_0$ is not stable unless His146 is protonated, providing an atomistic validation of the Perutz model. The possibility that extra large boxes are required to obtain meaningful results will have to be considered in evaluating existing and future simulations of a wide range of systems.