The phase separation of intrinsically disordered proteins is emerging as an important mechanism for cellular organization. However, efforts to connect protein sequences to the physical properties of condensates, that is, the molecular grammar, are hampered by a lack of effective approaches for probing high-resolution structural details. Using a combination of multiscale simulations and fluorescence lifetime imaging microscopy experiments, we systematically explored a series of systems consisting of diblock elastin-like polypeptides (ELPs). The simulations succeeded in reproducing the variation of condensate stability upon amino acid substitution and revealed different microenvironments within a single condensate, which we verified with environmentally sensitive fluorophores. The interspersion of hydrophilic and hydrophobic residues and a lack of secondary structure formation result in an interfacial environment, which explains both the strong correlation between ELP condensate stability and interfacial hydrophobicity scales, as well as the prevalence of protein-water hydrogen bonds. Our study uncovers new mechanisms for condensate stability and organization that may be broadly applicable.

Pancreatic K_ATP channel trafficking defects underlie congenital hyperinsulinism (CHI) cases unresponsive to the K_ATP channel opener diazoxide, the mainstay medical therapy for CHI. Current clinically used K_ATP channel inhibitors have been shown to act as pharmacochaperones and restore surface expression of trafficking mutants; however, their therapeutic utility for K_ATP trafficking-impaired CHI is hindered by high affinity binding, which limits functional recovery of rescued channels. Recent structural studies of K_ATP channels employing cryo-electron microscopy (cryoEM) have revealed a promiscuous pocket where several known K_ATP pharmacochaperones bind. The structural knowledge provides a framework for discovering K_ATP channel pharmacochaperones with desired reversible inhibitory effects to permit functional recovery of rescued channels. Using an AI-based virtual screening technology AtomNet followed by functional validation, we identified a novel compound, termed Aekatperone, which exhibits chaperoning effects on K_ATP channel trafficking mutations. Aekatperone reversibly inhibits K_ATP channel activity with a half-maximal inhibitory concentration (IC₅₀) ~9 μM. Mutant channels rescued to the cell surface by Aekatperone showed functional recovery upon washout of the compound. CryoEM structure of K_ATP bound to Aekatperone revealed distinct binding features compared to known high affinity inhibitor pharmacochaperones. Our findings unveil a K_ATP pharmacochaperone enabling functional recovery of rescued channels as a promising therapeutic for CHI caused by K_ATP trafficking defects.