NMR Determination of Protein pKa Values in the Solid State

Charged residues play an important role in defining key mechanistic features in many biomolecules. Determining the pK a values of large, membrane or fibrillar proteins can be challenging with traditional methods. In this study we show how solid-state NMR is used to monitor chemical shift changes during a pH titration for the small soluble β1 immunoglobulin binding domain of protein G. The chemical shifts of all the amino acids with charged side-chains throughout the uniformly- 13 C, 15 N-labeled protein were monitored over several samples varying in pH; pK a values were determined from these shifts for E27, D36, and E42, and the bounds for the pK a of other acidic side-chain resonances were determined. Additionally, this study shows how the calculated pK a values give insights into the crystal packing of the protein.