Atomistic modeling of macromolecular crowding predicts modest increases in protein folding and binding stability.

Theoretical models predict that macromolecular crowding can increase protein folding stability, but depending on details of the models (e.g., how the denatured state is represented), the level of stabilization predicted can be very different. In this study, we represented the native and denatured states atomistically, with conformations sampled from explicit-solvent molecular dynamics simulations at room temperature and high temperature, respectively. We then designed an efficient algorithm to calculate the allowed fraction, f, when the protein molecule is placed inside a box of crowders. That a fraction of placements of the protein molecule is disallowed because of volume exclusion by the crowders leads to an increase in chemical potential, given by Deltamu = -k(B)T lnf. The difference in Deltamu between the native and denatured states predicts the effect of crowding on the folding free energy. Even when the crowders occupied 35% of the solution volume, the stabilization reached only 1.5 kcal/mol for cytochrome b562. The modest stabilization predicted is consistent with experimental studies. Interestingly, a mixture of different sized crowders was found to exert a greater effect than the sum of the individual species of crowders. The stabilization of crowding on the binding stability of barnase and barstar, based on atomistic modeling of the proteins, was similarly modest. These findings have profound implications for macromolecular crowding inside cells.