Revealing hidden noncovalent forces of a disulfide bond in protein interactions and biomolecular condensation

Although disulfide bonds impose long-range geometric constraints that enhance protein stability, the existence of their noncovalent interactions remains uncertain. We investigated the dynamics and interactions of the intrinsically disordered protein (IDP) tau dGAE, which includes residue C322, using extensive NMR experiments. The enhanced transverse relaxation rates of amide- 15 N ( R 2 ) near the disulfide bond are primarily attributed to increased backbone rigidity. However, the long-range, disulfide-mediated weak interactions were clearly resolved even in excess water. These include disulfide-centered hydrogen bonds with NH groups (DCHB), and nonbonded interactions with the aromatic residue Y310 (DCNB). The operational Gibbs free energies (ΔG op ) of the combined DCHB and DCNB interactions, reflecting the redistribution from an ordered structured ensemble stabilized by these interactions to a more disordered IDP ensemble, were determined for the wild-type and Y310 mutants: 2.5, 1.5, and 3.0 kJ/mol for Y310, Y310N, and Y310W, respectively. Notably, the extent of liquid-liquid phase separation (LLPS) induced by these proteins correlated with their corresponding ΔG op values, which are counterbalanced by water interactions. DCHB and DCNB offer mechanistic insights not only into LLPS, but also into various biological processes, in which weak interactions can be significant. Disulfide-centered hydrogen bonds with NH groups (DCHB) and nonbonded interactions with Y310 (DCNB) in tau-dGAE are experimentally verified. The ΔG values of the wild-type and mutant proteins (1.5–3.0 kJ/mol) correlate precisely with their liquid–liquid phase separation tendency, offering insight into biological processes where weak interactions are critical. • Disulfide bonds mediate weak interactions beyond covalent constraints in water. • Disulfide-mediated H-bonds with HN and aromatic contacts are experimentally verified. • Interaction Gibbs free energies in water were determined to be 1.5–3.0 kJ/mol. • Weak disulfide-mediated forces play critical roles in tau dGAE LLPS.