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.
