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Toward the microscopic simulations of cell-like environments.

Periodic Reporting for period 1 - CROWDY (Toward the microscopic simulations of cell-like environments.)

Reporting period: 2019-05-01 to 2021-04-30

The interior of a living cell is a highly crowded place—in fact, up to 40 % of the entire volume is filled by proteins and other biomolecules. This means that a protein immersed in such a densely packed environment constantly bounces into other molecules and transiently binds to them before moving to other interaction partners.

The interactions with the crowded intracellular environment may affect various properties of proteins, such as their ability to move and the stability of their native conformations, that is, the three-dimensional structures that allow proteins to carry out their functions. Therefore, to understand how these vital molecules work inside cells, it is important to accurately describe the intracellular environment and its impacts on protein mobility and stability.

While it is often difficult to obtain experimental information on molecular interactions taking place in the extremely crowded and heterogeneous conditions, such microscopic details can be captured using computer simulations. However, the large spread of time- and length scales that are involved in such crowded systems poses a serious challenge to existing simulation techniques.

In this project, we implemented a multi-scale simulation scheme combining coarse-grained simulations (using the lattice Boltzmann molecular dynamics technique) with detailed atomistic simulations to investigate the diffusion and stability of proteins under conditions mimicking those existing in cells. This scheme allowed us to explore protein motions in large crowded systems and, subsequently, zoom in to investigate atomistic details of protein conformations and intermolecular interactions, including interactions with lipid membranes. The results revealed how crowding shapes protein stability and diffusion at different temperatures. Moreover, they provided microscopic insights into changes that occur in the cellular interior when a cell dies due to a high temperature.
In the course of the project, we considered crowded systems of varying complexity, ranging from solutions containing a single crowder species through a very heterogenous system mimicking the protein composition found in the Escherichia coli (E. coli) bacterium.

By examining the thermal stability of a model protein (chymotrypsin inhibitor 2) in different crowded environments, we showed that the stability effect of crowding depends on temperature. In particular, we found evidence of a crossover temperature below which the crowded environment destabilized the native state of the protein and above which it became stabilizing. We found that distinct interaction patterns with the crowders as well as the crowder size played a role in the balance between destabilization and stabilization (published in J. Phys. Chem. Lett. 2021).

We further investigated the diffusion, stability, and conformations of the barrels of superoxide dismutase 1 (SOD1), a protein whose unfolding has been implicated in the ALS disease. By analyzing the thermal unfolding of the protein, we identified the most fragile parts of the barrel and linked them to an increased interaction with the crowder. These analyses allowed us to rationalize why certain amino acid mutations on the SOD1 barrel can turn a destabilizing environment into a stabilizing one, an unexpected effect observed in experiments. Our findings highlighted the role of weak attractive interactions of a protein with its environment (published in J. Phys. Chem. Lett. 2020).

As the most complex system investigated during the project, we built a model mimicking a part of the E. coli interior. We used it to probe protein diffusion, contacts, and stability inside the heterogeneous protein mixture. In particular, we examined how protein motions and interactions are affected when parts of the E. coli interior become thermally unfolded. We correlated the results with findings from neutron scattering measurements to shed light on processes that accompany cell death.

Finally, we considered the diffusion of an enzyme (alpha-chymotrypsin) inside a model of a vesicle in the presence of crowders. Our simulations showed how crowders can modulate the proximity of the enzyme to the confining membrane, with possible impacts on the enzyme’s catalytic activity.
The newly-established simulation scheme combining a coarse-grained and an atomistic description of crowded systems opens the way for future investigations of various physiologically important proteins exposed to the crowded conditions in cells.

The obtained insights into the unfolding of the SOD1 barrel in cell-like environments may help understand its misfolding and aggregation linked to the ALS disease.

The information on temperature-dependent protein dynamics and stability in crowded environments will be a valuable contribution to our understanding of how living organisms adapt to different temperatures.

Overall, our findings underlined the importance of considering interactions with the crowded environment inside cells. Moreover, we demonstrated that molecular simulations can provide microscopic insights to rationalize experimentally observed crowding effects and to verify theoretical predictions.
Computational model of the E. coli cytoplasm
SOD1 barrel surrounded by thermally unfolded E. coli proteins