On the Influence of Trimethylamine-N-oxide (TMAO) and Pressure on Hydrophobic Interactions

Osmolytes are small organic molecules that influence the protein folding equilibrium and biomolecular condensates formed by liquid-liquid phase separation (LLPS). Thereby they can protect the cell from extracellular stress in the form of other molecules or external variables like pressure, temperature and pH, allowing life to flourish at extreme conditions. Trimethylamine-N-oxide (TMAO) is one such osmolyte, which has been studied due to its presence in deep sea organisms living in high pressure environments. Experimentally, it has been found that TMAO can counteract pressure denaturation and the disappearance of LLPS, both crucial in the functioning of the cell. However, there is still no consensus on the molecular mechanism governing the stabilising effect of TMAO and it is still highly controversial in the sense that it is not clear which interactions are responsible for TMAOs stabilising ability. Protein interactions can be manifold, ranging from electrostatic interactions (salt bridge formation between charged side chains), polar interactions, like hydrogen bonds, and hydrophobic interactions. Furthermore, the reason why TMAO is called a "piezolyte'', an osmolyte specialized in its pressure counteracting ability, and whether it is even distinct from other osmolytes at all is unsure. This work focuses on hydrophobic interactions, which are one of the main driving forces for protein folding and biocondensate formation. It proposes molecular mechanisms on the cumulative effect of TMAO and temperature, pressure or molecule size on hydrophobic interactions and hydrophobic hydration. For these studies a combination of structural analysis of the solvent and cosolute distribution, thermodynamic data and statistical mechanics analysis was used. Lastly, the folding equilibrium of a miniprotein has been analyzed to transfer the knowledge gained with hydrophobic molecules to a more complex system. A major problem of computational studies on TMAO effects is the existence of several force fields, which often can not reproduce experimental properties. The results in this work showed that the force field used is capable to capture the general experimentally observed trend of preferential TMAO binding to a small peptide. It is shown that preferential TMAO binding depends on temperature, solute size and charge state (protonation state) of the solute and its functional groups. Generally, the presence of charged groups contribute to TMAO depletion. TMAO is depleted (lowers the solubility of the solute) from small non-polar solutes at low temperatures and switches to preferential binding (increases the solubility of the solute) upon increasing the temperature. Furthermore, TMAO is depleted from small repulsive solutes at all temperatures, but preferentially binds to large repulsive solutes at ambient temperatures. For small solutes TMAO increases hydrophobic interactions independent of preferential TMAO binding, proving that TMAO can increase solute aggregation not only through a depletion mechanism, as is usually proposed in the literature for the stabilising TMAO effect, but also through preferential binding. Intriguingly, preferential TMAO binding to large repulsive solutes drives solute association through a surfactant-like mechanism, dominating at low TMAO concentrations. This leads to a non-monotonic trend in its effect on the association of non-polar solutes, as the contribution of attractive interactions drive solute dissociation, driving the equilibrium to the dissociated state at high TMAO concentrations. Furthermore, this non-monotonic trend prevails at high pressure, but the stabilising TMAO effect of the associated state is enhanced. This effect is due to the enhancement of the TMAO dipole moment upon pressure increase. Force fields which do not take this into account do not exhibit this effect. Thus, in the case of hydrophobic interactions TMAOs piezolytic abilities are caused by its increased dipole moment. Lastly, a protein having hydrophobic, charged and polar interactions was simulated. It has been shown that hydrophobic and electrostatic interactions are strengthened by the presence of TMAO. Additionally, the protein looses protein-solvent hydrogen bonds in the TMAO mixture compared to the pure water case. This destabilizes both the folded and unfolded protein state, but more so the unfolded state as it has a higher solvent accessible surface area. As a result, TMAO drives protein folding due to the loss of favorable protein-solvent hydrogen bonds. This thesis extends the current knowledge of TMAO effects on hydrophobic interactions. Furthermore, it takes collective effects into account, enhancing the knowledge across the temperature-pressure plane and adding knowledge about other functional group effects. Several stabilising effects of TMAO on hydrophobic interactions and protein folding have been discovered, serving to understand the manifold interactions of TMAO with more complex molecules. These findings can be used for a general understanding of cosolute effects on the polymer collapse equilibrium, protein folding equilibrium and the formation of biocondesates via liquid-liquid phase separation (LLPS)