Valid molecular dynamics simulations of human hemoglobin require a surprisingly large box size

Recent molecular dynamics (MD) simulations of human hemoglobin (Hb) give results in disagreement with experiment. Although it is known that the unliganded (T<span id="MathJax-Element-1-Frame" class="MathJax" style="position: relative;" tabindex="0" role="presentation" data-mathml=" 0"> 0 ) and liganded (R<span id="MathJax-Element-2-Frame" class="MathJax" style="position: relative;" tabindex="0" role="presentation" data-mathml=" 4"> 4 ) tetramers are stable in solution, the published MD simulations of T<span id="MathJax-Element-3-Frame" class="MathJax" style="position: relative;" tabindex="0" role="presentation" data-mathml=" 0"> 0 undergo a rapid quaternary transition to an R-like structure. We show that T<span id="MathJax-Element-4-Frame" class="MathJax" style="position: relative;" tabindex="0" role="presentation" data-mathml=" 0"> 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<span id="MathJax-Element-5-Frame" class="MathJax" style="position: relative;" tabindex="0" role="presentation" data-mathml=" 0"> 0 tetramer, to be manifested. Even in the largest box, T<span id="MathJax-Element-6-Frame" class="MathJax" style="position: relative;" tabindex="0" role="presentation" data-mathml=" 0"> 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.