Designing Protein Stabilizers: Small Molecule Effects on Protein Folding are Driven by Direct Interactions and Solvent Rearrangement

Proteins are marginally stable macromolecules, prone to denaturation when exposed to environmental stressors. This susceptibility burdens the development, storage, and transportation of biological therapeutics. Thus, biological formulations are generally accompanied with small molecule excipients – including amino acids and sugars – that stabilize the native state of proteins. While empirically these effects are well known, the molecular features of excipients that drive stability remain unclear. Additionally, due to the chemical heterogeneity of proteins, it is difficult to resolve contributions arising from many competing forces involved in folding. To establish a molecular picture, we have utilized molecular dynamics simulations to explore the effects of various excipient solutions on miniprotein folding. We observe that residue-additive interactions, along with perturbations of the water structure surrounding the miniproteins, are the primary determinants of stability. These results bring into focus the molecular motifs that act as determinants of stability, and provide insights towards protein folding manipulation via small molecule additives. The insights will provide molecular design principles to discover novel molecules for stabilizing biological therapeutics.