Proteins in vivo are immersed in a crowded environment of water, ions, metabolites, and macromolecules. In-cell experiments highlight how transient weak protein− protein interactions promote (via functional "quinary structure") or hinder (via competitive binding or "sticking") complex formation. Computational models of the cytoplasm are expensive. We tackle this challenge with an all-atom model of a small volume of the E. coli cytoplasm to simulate protein−protein contacts up to the 5 μs time scale on the specialpurpose supercomputer Anton 2. We use three CHARMM-derived force fields: C22*, C36m, and C36mCU (with CUFIX corrections). We find that both C36m and C36mCU form smaller contact surfaces than C22*. Although CUFIX was developed to reduce protein−protein sticking, larger contacts are observed with C36mCU than C36m. We show that the lifespan Δt of protein−protein contacts obeys a power law distribution between 0.03 and 3 μs, with ∼90% of all contacts lasting <1 μs (similar to the time scale for downhill folding).

Recent experimental data has shown that protein folding in the cytoplasm can differ from in vitro folding with respect to speed, stability, and residual structure. Here we investigate the all-atom molecular dynamics (MD) simulations of 9 copies of the model protein GTT WW domain in a small bacterial cytoplasm model using three force fields. GTT has been well-studied by MD in aqueous solution for comparison. We find that folded copies remain folded for up 25 μs, whereas unfolded copies do not fold for up to 190 μs. Unfolded GTT in our cytoplasm model does populate partly folded intermediates with one of the two hairpins formed. Relative to aqueous solution, GTT gets stuck in metastable states with a small RMSD and radius of gyration and extensive burial of surface area against other macromolecules. In particular, GTT is even able to form transient intermolecular β-sheets with other proteins, resulting in a “chimeric structure” that could be a precursor to oligomeric β-aggregates. We conclude that sticking, enhanced by the non-native mutations of GTT, is largely responsible, and we propose, on the basis of our result as well as recent experiments, that coevolution of protein surfaces with their solvation environment (including chaperones) is important for folding and diffusion of proteins in the cytoplasm.

How do enzymes form metabolons inside cells? To answer that question, we created an all-atom model of a section of the human cytoplasm and simulated it for over 30 microseconds. Among other proteins, nucleic acids, and metabolites, the model contains three successive members of the glycolytic cycle: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), and phosphoglycerate mutase (PGM). These enzymes interact to form transient, but long-lived, multi-enzyme complexes with characteristic lifetimes in the 1 to 5 μs range, thus modeling the functional metabolon structures that facilitate compartmentalization of metabolic pathways and substrate channeling in cell. We analyze the quinary structure between enzymes down to the formation of specific hydrogen-bonded interactions between side chains, together with the movement, in concert, of water molecules in or out between interacting amino acids to mediate contact formation and dissolution. We also observed large-scale enzymatic domain motion that has been proposed to convert between substrate-accessible and catalytically functional states: a direct hinge-bending motion of up to 28° changes the relative orientation of the N- and C- terminal domains of PGK, causing the initially open, and presumably inactive, conformation of PGK to sample both semi-closed and closed conformations. Although classical molecular dynamics (MD) cannot simulate enzymatic activity, closed structures are the functionally active forms of PGK, and their equilibrium with open structures opens the door for future quantum mechanics/molecular mechanics (QM/MM) and other reactive simulations of the cytoplasm.

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