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.