Mechanism of Thermal Protein Aggregation: Experiments and Molecular Dynamics Simulations on the High-Temperature...

Proteins that encounter unfavorable solvent conditions are prone to aggregation, a phenomenon that remains poorly understood. This work focuses on myoglobin (Mb) as a model protein. Upon heating, Mb produces amorphous aggregates. Thermal unfolding experiments at low concentration (where aggregation is negligible), along with centrifugation assays, imply that Mb aggregation proceeds via globally unfolded conformers. This contrasts studies on other proteins that emphasized the role of partially folded structures as aggregate precursors. Molecular dynamics (MD) simulations were performed to gain insights into the mechanism by which heat-unfolded Mb molecules associate with one another. A prerequisite for these simulations was the development of a method for generating monomeric starting structures. Periodic boundary condition artifacts necessitated the implementation of a partially immobilized water layer lining the walls of the simulation box. Aggregation simulations were performed at 370 K to track the assembly of monomeric Mb into pentameric species. Binding events were preceded by multiple unsuccessful encounters. Even after association, protein-protein contacts remained in flux. Binding was mediated by hydrophobic contacts, along with salt bridges that involved hydrophobically embedded Lys residues. Overall, this work illustrates that atomistic MD simulations are well suited for garnering insights into protein aggregation mechanisms.