Hydrogen-exchange behavior of the L20A mutant of the protein A B domain in guanidinium chloride: evidence for persistent native-like contacts.

Residual structure in unfolded proteins plays a crucial role in directing folding pathways. Here, we examined the unfolded state of the B domain of staphylococcal protein A carrying the L20A mutation, which disrupts native hydrophobic contacts between Leu20 and helix H3 (residues 41-56). The L20A mutant markedly reduced the thermodynamic stability of the native state. Using dimethylsulfoxide (DMSO)-quenched hydrogen/deuterium (H/D)-exchange NMR spectroscopy, we measured H/D-exchange kinetics for backbone amide protons in 6 M guanidinium chloride. At defined exchange times, the reaction mixture was rapidly transferred to a DMSO solution using a spin desalting column, thereby quenching exchange and allowing accurate NMR measurements. Even under the strongly denaturing condition of 6 M guanidinium chloride, the L20A mutant exhibited substantially lower protection against H/D exchange than the wild-type protein, particularly for residues in helix H3. These results indicate that native-like tertiary contacts between Leu20 and helix H3 persist in the unfolded ensemble and contribute to stabilizing residual structure. Our findings demonstrate that DMSO-quenched H/D-exchange NMR combined with targeted mutation provides a powerful approach for dissecting residual interactions and elucidating how early native-like contacts shape folding pathways.