NMR Determination of Protein p<i>K</i>_a Values in the Solid State

Charged residues play an important role in defining key mechanistic features in many biomolecules. Determining the p<i>K</i>_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 ¹³C,¹⁵N-labeled protein were monitored over several samples varying in pH; p<i>K</i>_a values were determined from these shifts for E27, D36, and E42, and the bounds for the p<i>K</i>_a of other acidic side-chain resonances were determined. Additionally, this study shows how the calculated p<i>K</i>_a values give insights into the crystal packing of the protein

Solid-State Characterization and Relative Formation Enthalpies To Evaluate Stability of Cocrystals of an Antidiabetic Drug

The current study integrates formation enthalpy and traditional slurry experiments to quickly assess the physical stability of cocrystal drug substance candidates for their potential to support drug development. Cocrystals of an antidiabetic drug (GKA) with nicotinamide (NMA), vanillic acid (VLA), and ethyl vanillin (EVL) were prepared and characterized by powder X-ray diffractometry (PXRD), spectroscopic, and thermal techniques. The formation enthalpies of the cocrystals, and their physical mixtures (GKA + coformer) were measured by the differential scanning calorimetry (DSC) method reported by Zhang et al. [Cryst. Growth Des. 2012, 12 (8), 4090−4097]. The experimentally measured differences in the relative formation enthalpies obtained by integrating the heat flow of each cocrystal against the respective physical mixture were correlated to the physical stability of the cocrystals in the solid state. The relative formation enthalpies of all of the cocrystals studied suggest that the cocrystals are not physically stable at room temperature versus their physical mixtures. To further address relative stability, the cocrystals were slurried in 30% v/v aqueous ethanol, and it was observed that all of the cocrystals revert to GKA within 48 h at room temperature. The slurry experiments are consistent with the relative instability of the cocrystals with respect to their physical mixtures suggested by the DSC results