Abstract
Despite extensive experimental and computational efforts to understand the nature of the hierarchy of protein fluctuations and the modulating role of the protein hydration shell, a detailed microscopic description of the dynamics of the protein-solvent system has yet to be achieved. By using single tryptophan protein phosphorescence, we follow site-specific internal protein dynamics over a broad temperature range and demonstrate three independent dynamic processes. Process I is seen at temperatures below the bulk solvent T(g), has low activation energy, and is likely due to fast vibrations that may be enabled by water mobility on the protein surface. Process II is observed above 170 K, with activation energy typical of β relaxations in a glass; it has the same temperature dependence as fluctuations of hydration shell waters. Process III is observed at T > 200 K; it has super-Arrhenius temperature dependence and closely follows the primary relaxation of the bulk. The fluorescence of pyranine bound to the protein reports on the mobility of water in the hydration shell; it reveals a shift in emission spectra with increasing temperature, indicative of a changing H-bond network at the surface of the protein. These results support a model of solvent-slaved protein dynamics.