Multi-mode water dynamics in hydration shells of villin headpiece subdomain protein in the solid state using deuterium and oxygen-17 NMR spectroscopy

Hydration shell properties in proteins remain an active topic of investigation due to their complexity and importance for biological processes. We focused on hydration shell dynamics in the solid state of the globular villin headpiece subdomain (HP36). We utilized H-2 (D2O hydration) and O-17 ((H2O)-O-17 hydration) solid-state NMR spectroscopy in combination with computational modeling to obtain a comprehensive picture of water motions, starting from high-amplitude modes such as diffusion and large-angle tetrahedral jumps and progressing to lower-amplitude modes such as 2-site deuteron flips and small-angle fluctuations. The measurements consisted of NMR line shapes as well as laboratory and rotating frame relaxation rates using novel approaches, conducted in the 300-170 K temperature range and at multiple values of magnetic field strengths. They permitted the precise determination of motional parameters such as fractions of different water layers, rate constants, and activation energies. Below about 250 K, both H-2 and O-17 longitudinal relaxation show clear non-exponential behaviors, with at least two components whose T-1 times differ by orders of magnitude. The water layer immediately adjacent to the protein surface remains mobile, as probed by the hydration dependence of NMR relaxation in the 20%-70% w/w water content range. Further, the observed non-exponentiality of O-17 T-1 rho relaxation at low temperatures suggests an exchange process between the layer adjacent to the protein and loosely bound shells. Based on prior results, we discuss correlations with dynamical changes in the hydrophobic core of HP36, thus obtaining insights into the interconnection of protein and water dynamics.