A Nonequilibrium Simulation Framework for Reproducing Protein Aggregation in All-Atom Molecular Dynamics Simulations

Protein aggregation is a central challenge in protein science and biopharmaceutical formulation, yet its reproduction by all-atom molecular dynamics (MD) simulations remains difficult within accessible computational limits. Here, we demonstrate that explicit control of the denaturation extent of initial protein structures enables reproducible thermal aggregation in MD simulations. Using hen egg white lysozyme (LYZ) as a model system, we generated unfolded conformational ensembles by high-temperature MD (300-700 K) and examined their aggregation behavior. Two factors proved critical for reproducing LYZ aggregation: a sufficiently high initial denaturation extent and direct initiation of simulations without an additional structural relaxation step. Under these conditions, 100 ns simulations successfully reproduced experimentally established additive effects: arginine suppressed aggregation only at high concentration (1 M), while sodium chloride promoted aggregation via nonspecific ionic effects. This proof-of-concept study establishes denaturation-controlled MD as a practical framework for computational evaluation of protein aggregation and stabilizer effects.