Hydration water dynamics in bovine serum albumin at low temperatures as studied by deuterium solid-state NMR

Solid state 2H NMR was used to investigate changes in the structure and dynamics of hydration waters of bovine serum albumin (BSA) due to glass transitions. The 2H NMR spectra were separated into fast and slow components based on differences in spin-lattice relaxation time T1. The fast components corresponded to water molecules interacting with protein while the slow components were the water molecules similar to bulk water and deuterons of the protein backbone. Simulation analysis of the 2H NMR spectra of the fast components was used to assess the mode and rate of motions of hydration waters around the protein. At low temperatures, the water molecules underwent a 180° flip and slow reorientation in the tetrahedral sites. The distribution of the rate of the 180° flip and the D-O-D angle of water molecules were clarified. The distribution of the D-O-D angle of water molecules spread with decreasing temperature. The marked slowing down in the reorientation of water molecules was observed at a glass transition of around 200 K, which is linked to the disordered region of the protein. In contrast, the 180° flip of water molecules occurred frequently, even below 200 K. A freeze of the 180° flip of water molecules was observed around the glass transition temperature of 110 K, where primary hydrate water formed a direct hydrogen bond with the protein, making it perfectly immobile.