Are Current Atomistic Force Fields Accurate Enough to Study Proteins in Crowded Environments?

The high concentration of macromolecules in the crowded cellular interior influences different thermodynamic and kinetic properties of proteins, including their structural stabilities, intermolecular binding affinities and enzymatic rates. Moreover, various structural biology methods, such as NMR or different spectroscopies, typically involve samples with relatively high protein concentration. Due to large sampling requirements, however, the accuracy of classical molecular dynamics (MD) simulations in capturing protein behavior at high concentration still remains largely untested. Here, we use explicit-solvent MD simulations and a total of 6.4 µs of simulated time to study wild-type (folded) and oxidatively damaged (unfolded) forms of villin headpiece at 6 mM and 9.2 mM protein concentration. We first perform an exhaustive set of simulations with multiple protein molecules in the simulation box using GROMOS 45a3 and 54a7 force fields together with different types of electrostatics treatment and solution ionic strengths. Surprisingly, the two villin headpiece variants exhibit similar aggregation behavior, despite the fact that their estimated aggregation propensities markedly differ. Importantly, regardless of the simulation protocol applied, wild-type villin headpiece consistently aggregates even under conditions at which it is experimentally known to be soluble. We demonstrate that aggregation is accompanied by a large decrease in the total potential energy, with not only hydrophobic, but also polar residues and backbone contributing substantially. The same effect is directly observed for two other major atomistic force fields (AMBER99SB-ILDN and CHARMM22-CMAP) as well as indirectly shown for additional two (AMBER94, OPLS-AAL), and is possibly due to a general overestimation of the potential energy of protein-protein interactions at the expense of water-water and water-protein interactions. Overall, our results suggest that current MD force fields may distort the picture of protein behavior in biologically relevant crowded environments.Author Summary: Protein behavior is strongly affected by highly crowded and interaction-rich environments, i.e., typical conditions in both biologically relevant systems, such as the cellular interior, and solution-based structural experiments, including NMR and different spectroscopies. On the other hand, primarily because of limited computational power, molecular dynamics (MD) simulations, a premier high-resolution method for analyzing structure, dynamics and interactions of proteins, have been predominantly used to study individual proteins at infinite dilution. To fill this gap, we use MD simulations to study the behavior of wild-type (aggregation-resistant) and oxidatively damaged (aggregation-prone) forms of villin headpiece at high concentration, and reveal unexpected limitations and inaccuracies of modern-day MD force fields when it comes to modeling proteins at physiologically or experimentally relevant concentrations.