Experimentally test predictions of folding theory and MD simulations in cells

Recent in-cell studies have demonstrated that classic in vitro assays do not reproduce cellular protein or RNA interactions. Furthermore, differences between in-cell and in vitro cannot be easily predicted. While inert macromolecules have traditionally been used to mimic the stabilizing effect of cellular crowding, they do not account for transient sticking interactions between molecules in cells that can be stabilizing or destabilizing. We believe that a simple mixture could be used to study a variety of cytoplasmic protein interactions, such as folding, binding and assembly, and enzymatic reactions. Indeed, our mixture is not limited to mimicking protein interactions; At Yale, we have determined that the same mixture can be used to reproduce RNA and peptide folding behaviors in vitro. Improving our knowledge of in-cell ‘folding principles’ will make it possible to quantitatively assess MD simulations and thus formulate truly predictive in vivo protein models.