Buried Ion-Pairs Reshape Folding Pathways of Artificial Proteins

Ion-pairs are central charged motifs that control the biological function of several proteins. Yet, the physical principles linked to the formation of ion-pairs during protein folding remain poorly understood. Here, we show that buried ion-pairs of de novo-designed helical bundle proteins reshape the folding pathways by introducing kinetic barriers that are overcome by a hydrophobic collapse linked to packing of the protein core. By combining protein design with co-translational folding studies, nuclear magnetic resonance (NMR) spectroscopy, and molecular dynamics simulations, we show that introduction of a buried ion-pair leads to a shifted folding transition that balances desolvation and electrostatic interactions within the protein and the bulk solvent. Moreover, we find that the folding pathway is sensitive to modifications in non-polar interactions and motifs that electrostatically shield the ion-pair from its surroundings. Our combined findings provide a blueprint for understanding how functional motifs affect protein folding and how mutation of charged networks alter the stability of proteins.