High-pH NMR to Identify Macromolecular Hydrogen-Bonds and Foldons

O. For two α-helical coiled coils and three globular proteins, we performed alkaline unfolding experiments taking advantage of amide NMR signal attenuation from unstructured segments. Increasing the pH led to a progressive loss of native amide proton NMR signals, revealing an unfolding hierarchy where "foldons" remaining at the highest pH had the most persistent H-bonds under EX1 exchange conditions. The foldons observed at high pH are consistent with partially folded structures previously characterized near neutral pH by native state hydrogen exchange, equilibrium unfolding, and protein fragment studies. For β-sheet proteins, foldons correspond to regions with high inter-residue contact density, whereas in coiled coils they demarcate regions with high α-helical propensity. High-pH NMR experiments provide a sensitive, fast, and broadly applicable approach to map H-bonding. Additionally, they offer the opportunity to explore uncharted protein dynamics and unfolding pathways under basic pH conditions.