Multimolecule test-tube simulations of protein unfolding and aggregation

Abstract

Molecular dynamics simulations of protein folding or unfolding, unlike most in vitro experimental methods, are performed on a single molecule. The effects of neighboring molecules on the unfolding/ folding pathway are largely ignored experimentally and simply not modeled computationally. Here, we present two all-atom, explicit solvent molecular dynamics simulations of 32 copies of the Engrailed homeodomain (EnHD), an ultrafast-folding and -unfolding protein for which the folding/unfolding pathway is well-characterized. These multimolecule simulations, in comparison with single-molecule simulations and experimental data, show that intermolecular interactions have little effect on the folding/unfolding pathway. EnHD unfolded by the same mechanism whether it was simulated in only water or also in the presence of other EnHD molecules. It populated the same native state, transition state, and folding intermediate in both simulation systems, and was in good agreement with experimental data available for each of the three states. Unfolding was slowed slightly by interactions with neighboring proteins, which were mostly hydrophobic in nature and ultimately caused the proteins to aggregate. Protein-water hydrogen bonds were also replaced with protein-protein hydrogen bonds, additionally contributing to aggregation. Despite the increase in protein-protein interactions, the protein aggregates formed in simulation did not do so at the total exclusion of water. These simulations support the use of single-molecule techniques to study protein unfolding and also provide insight into the types of interactions that occur as proteins aggregate at high temperature at an atomic level.protein folding | protein dynamics T he folding pathway of the Engrailed homeodomain (EnHD) has been extensively characterized through a combination of experimental and computational techniques. EnHD is a threehelix bundle protein, with helices I and II packing antiparallel and helix III docking across them (Fig. 1). EnHD folds and unfolds on ultrafast timescales, which makes its folding pathway a good candidate to be studied by simulation. The structure of the transition state (TS) was first predicted by molecular dynamics (MD) simulations (1) and later validated by experimental techniques (2). A folding intermediate was identified by experiment (3) and structurally characterized by MD (4), and the MD-predicted structure was later validated by NMR (5). In addition, the protein adheres to the principle of microscopic reversibility in simulations at its melting temperature where unfolding and refolding occur in a single continuous trajectory (6). Similarly, when high-temperature unfolding simulations are quenched to folding permissive temperatures, the protein refolds by the reverse of unfolding (7). Despite the fact that the experimental techniques used provide ensemble measurements and the MD simulations are single-molecule (SM) in nature, the agreement has been very good, allowing for a much richer description of the folding/unfolding process of EnHD ...

Spatial Heat Maps from Fast Information Matching of Fast and Slow Degrees of Freedom: Application to Molecular Dynamics Simulations

“…(It is interesting to note that McCully et al 20 simulated a multi-molecular system consisting of 32 copies of this protein. They compared this with 10 single-molecule simulations, the one we are using here being the #2 among those 10.)…”

Structural Heterogeneity and Dynamics of the Unfolded Ensemble

“…This is analogous to the "induced fit" versus "conformational selection"controversy in protein allostery, which has a long history. [54][55][56] In a first approach, by simulating the unfolding of a small ensemble of a ultrafast-folding and -unfolding protein (32 copies of the engrailed homeodomain), McCully et al [57] determined that the presence of neighboring protein molecules does not significantly alter the unfolding pathway, but does affect its kinetics by slowing down the process. Some examples of proteins that maintain various degrees of disorder in the bound state have been described in the last few years, [47] shedding light on the possibility of having a continuous structural spectrum of functional proteins.…”

Using simulations to provide the framework for experimental protein folding studies

“…However, results depend significantly upon the size of the crowders and are non-additive for combinations of different crowding agents, pointing out to the necessity of using composite mixtures to mimic reality both in silico and in vitro . A recent study directly addressed the effect of neighboring molecules on native state dynamics and thermal unfolding for a well-characterized system [64]. In comparing these “test-tube” simulations (18 mM protein) to conventional single-molecule simulations it was found that neighboring molecules slowed unfolding slightly but the pathway was unaffected.…”