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Networks of electrostatic and hydrophobic interactions modulate the complex folding free energy surface of a designed βα protein

Significance Natural protein sequences have evolved to fold efficiently to their functional forms, traversing a relatively smooth energy surface with minimal frustration. The optimization of stability in de novo-designed proteins, without regard to dynamical processes required for function, can have unintended consequences on energy surfaces. Electrostatic and hydrophobic networks of side chains for Di-III_14, a small βα protein with a natural topology, create a rough energy surface whose high-energy states reflect the progressive unlocking of a tightly packed interior. Unbiased explorations of sequence space can reveal complex properties not seen in natural proteins, expanding our understanding of the relationships among sequence, structure, folding, and function of these ubiquitous biopolymers. The successful de novo design of proteins can provide insights into the physical chemical basis of stability, the role of evolution in constraining amino acid sequences, and the production of customizable platforms for engineering applications. Previous guanidine hydrochloride (GdnHCl; an ionic denaturant) experiments of a designed, naturally occurring βα fold, Di-III_14, revealed a cooperative, two-state unfolding transition and a modest stability. Continuous-flow mixing experiments in our laboratory revealed a simple two-state reaction in the microsecond to millisecond time range and consistent with the thermodynamic results. In striking contrast, the protein remains folded up to 9.25 M in urea, a neutral denaturant, and hydrogen exchange (HDX) NMR analysis in water revealed the presence of numerous high-energy states that interconvert on a time scale greater than seconds. The complex protection pattern for HDX corresponds closely with a pair of electrostatic networks on the surface and an extensive network of hydrophobic side chains in the interior of the protein. Mutational analysis showed that electrostatic and hydrophobic networks contribute to the resistance to urea denaturation for the WT protein; remarkably, single charge reversals on the protein surface restore the expected urea sensitivity. The roughness of the energy surface reflects the densely packed hydrophobic core; the removal of only two methyl groups eliminates the high-energy states and creates a smooth surface. The design of a very stable βα fold containing electrostatic and hydrophobic networks has created a complex energy surface rarely observed in natural proteins.